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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Dev Biol. 2011 Feb 23;353(1):61–71. doi: 10.1016/j.ydbio.2011.02.018

The Notch effector gene Hes1 regulates migration of hypothalamic neurons, neuropeptide content and axon targeting to the pituitary

Paven K Aujla, Adriana Bora, Pamela Monahan, Jonathan V Sweedler, Lori T Raetzman *
PMCID: PMC3077720  NIHMSID: NIHMS283319  PMID: 21354131

Abstract

Proper development of the hypothalamic-pituitary axis requires precise neuronal signaling to establish a network that regulates homeostasis. The developing hypothalamus and pituitary utilize similar signaling pathways for differentiation in embryonic development. The Notch signaling effector gene Hes1 is present in the developing hypothalamus and pituitary and is required for proper formation of the pituitary, which contains axons of arginine vasopressin (AVP) neurons from the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). We hypothesized that Hes1 is necessary for the generation, placement and projection of AVP neurons. We found that Hes1 null mice show no significant difference in cell proliferation or death in the developing diencephalon at embryonic day 10.5 (e10.5) or e11.5. By e16.5, AVP cell bodies are formed in the SON and PVN, but are abnormally placed, suggesting that Hes1 may be necessary for the migration of AVP neurons. GAD67 immunoreactivity is ectopically expressed in Hes1 null mice, which may contribute to cell body misplacement. Additionally, at e18.5 Hes1 null mice show continued misplacement of AVP cell bodies in the PVN and SON and additionally exhibit abnormal axonal projection. Using mass spectrometry to characterize peptide content, we found that Hes1 null pituitaries have aberrant somatostatin (SS) peptide, which correlates with abnormal SS cells in the pituitary and misplaced SS axon tracts at e18.5. Our results indicate that Notch signaling facilitates the migration and guidance of hypothalamic neurons, as well as neuropeptide content.

Keywords: hypothalamus, development, notch, Hes1, AVP, SS, PVN, SON

INTRODUCTION

The hypothalamic-pituitary axis (HP) is a master controller of endocrine processes, such as metabolism, growth and reproduction. The neuroendocrine hypothalamus contains nuclei with two distinct neuronal populations: magnocellular and parvocellular. Magnocellular neurons located in the paraventricular nuclei (PVN) and the supraoptic nuclei (SON) release arginine vasopressin (AVP) and oxytocin (OT) from their axonal terminals within the posterior lobe (PL) of the pituitary. OT acts on the periphery by inducing lactation and uterine contraction. In the central nervous system, OT facilitates social behavior, including parental care and bonding (Lim, Bielsky, Young. 2005; Lim, Young. 2006). AVP also exerts central effects on behavior, including aggression (Heinrichs, von Dawans, Domes. 2009). AVP released from the PL is crucial to nutrient reabsorption and regulating the body’s response to stress.

The hypothalamic anterior periventricular (aPV) nucleus contains parvocellular somatostatin (SS)-releasing neurons, and growth hormone-releasing hormone (GHRH) neurons are found in the arcuate nucleus (ARN). GHRH activates secretion of somatotropes in the anterior lobe of the pituitary (AL). Somatotropes secrete growth hormone (GH) and promote linear growth and metabolism, and SS inhibits the secretion of GH to regulate this process.

Magnocellular neurons, parvocellular neurons and pituitary cells demonstrate striking temporal and spatial coordination in events that regulate their differentiation. These cells form from the embryonic basal plate, with the anterior ridge generating the AL, and the adjacent region developing into the hypothalamus and the PL (Couly, Douarin. 1987, Couly, Douarin. 1988, Kawamura, Kikuyama. 1995, Kouki et al. 2001). In the mouse, hypothalamic neurons are generated between e10.5 and e12.5 from the proliferative neuroepithelium of the third ventricle (Shimada, Nakamura. 1973). Several signaling pathways and transcription factors have been implicated in formation of these neurons in the PVN and SON (Michaud, et al. 1998; Michaud, et al. 2000; Hosoya, et al. 2001). From the neuroepithelium, neurons migrate laterally to form the SON, or medially to form the PVN and aPV by e14.5 (Altman, Bayer. 1978; Altman, Bayer. 1979; Bayer, Altman. 1987). Various guidance cues, specifically, members of the Netrin, Slit/Robo and Semaphorin/Plexin/Neuropilin families are expressed in and around the developing PVN and SON, and have been implicated in cell migration (Deiner, Sretavan. 1999; Xu, Fan. 2007; Xu, Fan. 2008). GABAergic neurons have also been implicated in proper boundary formation of the PVN and VMN regions of the hypothalamus during development through GABAA and GABAB receptors (Dellovade, et al. 2001; Davis, Henion, Tobet. 2002; McClellan, Calver, Tobet. 2008; McClellan, Stratton, Tobet. 2010).

Although initial studies have begun to identify factors that are generally required for formation and migration of hypothalamic neurons, the mechanisms regulating specific neuroendocrine cell development remain unclear. We hypothesize that the Notch signaling pathway is required for proper formation, migration and projection of AVP and SS neurons to the pituitary.

Notch signaling is an evolutionarily conserved mechanism that guides progenitor maintenance and cell specification in the developing nervous system. In the cerebellum, loss of Notch1 results in the premature differentiation of neurons at the expense of undifferentiated cells (Lutolf, et al. 2002), and persistent activation of Notch2 maintains precursors in a proliferative state (Solecki, et al. 2001). Similarly, Hes1 and Hes3 double null mice show premature neuron formation in the mid/hind brain and subsequent loss of midbrain and anterior hindbrain structures (Hirata, et al. 2001). Additionally, overexpression of Hes1 in the telencephalon inhibits neuronal differentiation (Ohtsuka, et al. 2001).

Previous studies have delineated the importance of the Notch pathway in early specification events that regulate cell fate in the developing HP. The Notch effector gene Hes1 is spatially and temporally restricted to the developing diencephalon and pituitary during development, and its expression must be silenced for pituitary cell differentiation to occur (Zhu, et al. 2006; Kita, et al. 2007; Raetzman, Cai, Camper. 2007). Hes1 null mice survive until embryonic day 18.5 (e18.5) and show a reduction in PL size (Raetzman, Cai, Camper. 2007; Himes, Raetzman. 2009), which contains terminal axons of AVP and OT neurons. Previous studies have shown that the development of the ventral diencephalon (VD) also relies on Hes1, with Hes1 null mice displaying hypoplastic phenotypes of the developing pituitary and diencephalon (Akimoto, et al. 2010). Importantly, there is emerging evidence implicating Hes1 in cell migration and cell placement in the developing pituitary (Himes, Raetzman. 2009). Given that the primordial pituitary and hypothalamus share signaling pathways that generate differentiation and migration cues, it is likely that Hes1 may play a role in the development and placement of endocrine neurons within the hypothalamus.

In order to address the extent that Notch signaling is required for functional neuronal development within the HP, we analyzed Hes1 null mice and control littermates at various stages of embryonic development. We found no significant difference in the number of proliferating cells or cell death in the VD. AVP cell bodies are specified in Hes1 null mice and aberrantly placed, which correlates with ectopic GAD67 expression within these regions. We found abnormal projections of AVP-positive axons to the PL in Hes1 null mice. Additionally, we utilized mass spectrometry-based peptidomics (Li, Sweedler. 2008) to screen for peptide alterations in Hes1 null pituitaries at e18.5. We uncovered AVP-related products in Hes1 null mice as well as an unexpected SS peptide content. Further analyses showed a reduction of SS-positive cells in the aPV, aberrant SS-positive axons, and abnormal SS-positive expression in the PL of Hes1 null mice. The alterations in peptide content, and axon pathfinding to and termination in the pituitaries of Hes1 null mice indicate that Notch signaling facilitates the formation of AVP and SS neurons, guidance of hypothalamic axons to the pituitary, and neuropeptide processing.

MATERIALS AND METHODS

Animals

Hes1 mutant mice were previously generated by replacing the first 3 exons with a neomycin-resistance cassette (Ishibashi, et al. 1994). Breeding colonies were generated at the University of Illinois at Urbana-Champaign (UIUC) and maintained on a mixed genetic background of C57B1/6J and CD1 mice. All animal procedures were approved by the UIUC Institutional Animal Care and Use Committee. Heterozygous males and females were mated to generate mixed genotype litters, which were genotyped as previously described (Jensen, et al. 2000). Embryos were collected at e10.5, e11.5, e16.5 and e18.5, and either prepared for immunohistochemistry or peptide extraction at e18.5.

Immunohistochemistry

After collection, embryos were fixed in formaldehyde, embedded in paraffin and processed for immunohistochemistry as previously described (Monahan, Rybak, Raetzman. 2009). Primary antibodies were as follows: rabbit anti-arginine vasopressin (Abcam, Cambridge, MA USA 1:500; Fitzgerald Industries, Acton, MA, USA 1:500), rabbit anti-somatostatin-28 antibodies (Bachem, Torrance, CA, USA 1:500; Millipore, Billerica, MA, USA 1:100), rabbit anti-phosphohistone H3 (PH3) (Millipore, 1:300). Species specific secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA, USA). Nickel (II) sulfate 3,3-diaminobenzidine (NiDAB) immunohistochemistry, adapted from (Kramer, et al. 2005) was performed on parasagittal sections embedded at a 30° horizontal plane. Subsequent to primary and secondary antibody incubation, a Vectastain kit (Vector Laboratories, Burlingame, CA, USA) diluted in PBS was used. Slides were then equilibrated in 0.175M sodium acetate. Next, NiDAB solution (2.5% nickel II sulfate, 2% DAB, 0.02% H202 in sodium acetate) was applied to the slides for 20–30 minutes at room temperature. Slides were then washed in 0.175M sodium acetate 2 times for 5 minutes and PBS 2 times for 5 minutes. Finally, slides were counterstained with methyl green, dehydrated and mounted with Permount (Fisher). Samples were visualized at a 100×, 200× and 400× magnification using a Leica DM 2560 microscope. Images were taken using Q Capture Pro software (QImaging, Surrey, BC, Canada) and processed with Adobe Photoshop, version 11.0.2 (Adobe Systems Incorporated, San Jose, CA, USA).

PH3-positive and AVP-positive cell quantification

Midsagittal sections of the VD taken from Hes1 null embryos and littermate controls at e10.5 and e11.5 were immunostained with PH3 and DAPI as described. Sections from the mid-PVN and mid-SON of Hes1 null embryos and littermate controls at e16.5 were immunostained with AVP as described. Images were taken at 200× magnification. The number of solid PH3-positive cells in the VD were counted and divided by the total number of DAPI-positive cells in the VD to obtain the proportion of PH3-positive cells in the VD. The numbers of AVP-positive cells were counted in the mid-PVN and mid-SON. For PH3 and AVP cell counts, 4 sections per animal were analyzed and the average proportion of PH3-positive or AVP-positive cells was compared between three Hes1 null embryos and three littermate controls. These values were tested for statistical significance using a Student’s t test (SAS 9.1 software; SAS Institute, Cary, NC, USA).

Quantitative real time-PCR

Whole brains were dissected at e16.5 and snap frozen in ethanol. RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed to create cDNA from 0.5g of isolated mRNA template. qRT-PCR was performed using cDNA with primers for AVP, GAD1 and GAPDH. The primer sequences are as follows: AVP forward 5′ CTC TCC GCT TGT TTC CTG AG 3′, AVP reverse 5′ CTC TTG GGC AGT TCT GGA AG 3′, GAD1 forward 5′ CTC CAA GGA TGC AAC CAG AT 3′, GAD1 reverse 5′ CTG GAA GAG GTA GCC TGC AC 3′, GAPDH forward 5′ GGT GAG GCC GGT GCT GAG TAT G 3′, GAPDH reverse 5′ GAC CCG TTT GGC TCC ACC CTT C 3′. Samples were run and analyzed on Bio-Rad iCycler IQ (Bio-Rad Laboratories, Hercules, CA, USA). The PCR conditions for all primer sets used were 95 C for 20 sec, 55 C for 30 sec, and 72 C for 30 sec (40 cycles). AVP and GAD1 cycle threshold was normalized to GAPDH cycle threshold and data was analyzed with the ΔCT method.

Peptide extraction for mass spectrometry

Pituitaries from embryos were isolated and peptides were extracted from the isolated pituitaries using acidified acetone. Pituitaries from were pooled and analyzed with liquid chromatography mass spectrometry (LC-MS) to determine the sequences of the major peptides. Individual pituitaries from two control and two Hes1 null mice were analyzed with direct matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS (see Supplemental methods).

Peptide identifications via liquid chromatography mass spectrometry

Chromatographic separation of the extracted peptides was achieved using a CapLC system (Micromass, UK). The samples were loaded onto a trap column and eluted onto a reversed phase capillary column. For LC-MALDI-TOF/TOF-MS analysis, chromatographic separation was performed and chromatographic elutant spotted onto a MALDI plate. A Bruker Ultraflex II TOF/TOF instrument was used to analyze each sample.

RESULTS

Loss of Hes1 does not alter progenitor proliferation in the early developing ventral diencephalon

Generation and proliferation of neuronal precursors that populate the endocrine SON and PVN occurs between e10.5 and e12.5 in mouse (Karim, Sloper. 1980). We hypothesize that Hes1 is needed to control proliferation of VD progenitors during early embryonic development. To assess whether proliferating VD progenitors are completing mitotic division, we utilized PH3 as a marker of the M and late G2 phase of the cell cycle. In controls, proliferating PH3-positive cells are detected in the VD of e10.5 animals (Fig. 1A, arrows). In comparison, Hes1 null animals appear to have similar expression patterns, with cells in mitosis lining the ventricular zone (Fig. 1B). Cell count analysis of VD progenitors reveals no significant changes in proliferation between control (mean percent 16.8±1.6; n=3) and Hes1 null animals (mean percent 17.6±0.8; n=3). By e11.5, controls show VD expansion, with cells in mitosis remaining in the ventricular zone (Fig. 1C). Hes1 null VD proliferation again mirrors that of controls (Fig. 1D) and cell count analysis reveal no significant change in the numbers of cells in mitosis between control (mean percent 15.2±0.8; n=3) and Hes1 null (mean percent 13.9±1.2; n=3) animals. These data taken together indicate that loss of Hes1 does not significantly alter VD progenitor proliferation, specifically with regard to the population of cells in mitosis, and that Hes1 alone may not contribute to expansion of progenitors at early stages of VD development.

Fig. 1. Loss of Hes1 does not alter progenitor proliferation or cell death in the early developing ventral diencephalon.

Fig. 1

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Midsagittal sections of mouse embryos at e10.5 and e11.5 were immunostained with phosphohistone H3 (PH3) to visualize proliferating progenitors in the developing ventral diencephalon (VD). At e10.5, PH3-positive cells are detected in the ventricular zone of the VD in both controls (A, arrows) and Hes1 null (B, arrows) animals. At e11.5, like e10.5, proliferating progenitors are restricted to the ventricular zone in both control (C) and Hes1 null animals (D). Cell death was detected using TUNEL in e10.5 and e11.5 control and Hes1 null animals. At e10.5, few cells are undergoing cell death in the VD in control (E, arrows) or Hes1 null (F, arrows) animals. At e11.5 few if any dying cells can be visualized in the VD of both control (G, arrows) and Hes1 null (H, arrows) animals. Two sections from three individual embryos were examined for each genotype. Scale bar indicates 50 μm.

During pituitary development, loss of Hes1 can induce cell death in progenitors that have prematurely exited the cell cycle (Raetzman, Cai, Camper. 2007). We hypothesize that loss of Hes1 in the developing VD may also cause loss of progenitors through cell death. In order to assess cell death in the VD, we utilized TUNEL immunohistochemistry to label cell populations undergoing cell death during early development. At e10.5, controls (Fig. 1E, arrows) and Hes1 null (Fig. 1F, arrows) animals reveal few TUNEL positive cells in the developing VD. At e11.5, few if any cells in the VD have positive staining in either the controls (Fig. 1G) or Hes1 null (Fig. 1H) animals. This lack of TUNEL-positive cells indicates that loss of Hes1 does not induce cell death in the growing VD.

Hes1 is necessary for proper formation of AVP neurons within the SON and PVN

Expression studies have discovered that AVP neurons within the PVN and SON are formed by e14.5 in the mouse (Altman, Bayer. 1978; Altman, Bayer. 1979; Bayer, Altman. 1987). However, the precise mechanisms underlying cell placement and axon migration of neuroendocrine hypothalamic neurons remain elusive. We hypothesize that the Notch signaling is important for proper formation and migration of hypothalamic neurons that project to the pituitary.

In both Hes1 null and control mice at e16.5, the SON is formed in a lateral cluster superior to the optic nerve, and AVP-positive cells migrating to the SON can be visualized (Fig. 2). In control animals, at the level of the PL, there are no AVP-positive cells, as this region is posterior to the SON (Fig. 2A, 2A′). However, Hes1 null animals display a lateral cluster as well as a ventromedial cluster of AVP positive cells at the level of the PL (Fig. 2B, 2B′), suggesting that SON boundaries reach more posteriorly in Hes1 null animals. At the more anterior level of the median eminence (ME), control animals show AVP-positive cell bodies superior to the optic nerve, as well as a ventromedial cluster, medial to the SON (Fig. 2C, 2C′). In contrast, Hes1 null animals show a more dispersed SON boundary, as well as a medial cluster of cells, which span more dorsally compared to control AVP-positive cells (Fig. 2D, 2D′). In more anterior sections, the number of AVP neurons in the SON is comparable between Hes1 null and control animals (data not shown). There is no significant difference in relative levels of AVP mRNA in whole brains at e16.5 between control and Hes1 null animals (Supplementary Fig. 1), indicating that loss of Hes1 alters cell body position but not overall AVP neuron number within the PVN and SON.

Fig. 2. Loss of Hes1 causes increased and aberrant GABAergic neuron expression and faulty placement of AVP neurons in the SON and PVN at e16.5.

Fig. 2

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A. Represents area within red box of A′ and shows no immunohistochemical detection of arginine vasopressin (AVP) cell bodies in coronal sections at the level of the PL in controls. B. Represents area within red box of B′ and shows that at the same level of the PL, Hes1 null mice show AVP-positive cells in a medial cluster as well as a few scattered cells laterally. C. Represents area within the red box of C′ at the level of the medial median eminence (ME), 198μm anterior to the PL, and shows medial AVP-positive cell bodies in a ventromedial cluster as well as a cluster superior to the optic nerve in the supraoptic nucleus (SON) in controls. D. Represents area within the red box of D′ and shows more dorsal AVP-positive cells at the medial median eminence (ME), 126μm anterior to the PL in Hes1 null animals. Control animals show GAD67 immunoreactivity around the region of the SON at all levels, but not within the SON (E, red box). In contrast, Hes1 null animals display an increase of GAD67-positive cells in and around the SON (F, red box denotes SON). Control animals show AVP immunoreactivity in the paraventricular nucleus (PVN), forming a trapezoid pattern at the third ventricle (G), while Hes1 null animals have more diffuse AVP-positive cells in that region (I). GAD67-positive cells mostly surround the PVN in control animals (H), but are found within the PVN in Hes1 null mice. Fifty sections from 6 individual embryos were examined for each genotype. Scale bar indicates 50μm.

GABAergic neurons have been shown to surround the PVN during development in order to establish the boundaries of AVP neurons within this region (McClellan, Stratton, Tobet. 2010). We therefore examined the expression of GAD67, which catalyzes the conversion of glutamate to GABA and marks GABAergic neurons, to assess if loss of Hes1 affects the expression of GABAergic neurons surrounding the developing SON and PVN at e16.5. In normal animals, GAD67 is expressed surrounding the SON, such that GAD67 immuno-negative regions delineate the SON region (Fig. 2E). In contrast, Hes1 null animals display GAD67 expression within the SON and appear to have increased GAD67-immunopositive neurons at the level of the SON (Fig. 2F).

The boundary of AVP neurons within the PVN is also affected in Hes1 null animals at e16.5. AVP-positive cells form a distinct trapezoid shape in control animals at e16.5 (Fig. 2G), but AVP expression is unrestricted around the third ventricle of Hes1 null littermates (Fig. 2I). Hes1 littermate controls show GAD67 immunoreactivity around the PVN, but little expression within this region (Fig. 2H). In contrast, GAD67 immunoreactivity appears increased at the level of the PVN in Hes1 null animals, and is present within the PVN region (Fig. 2J). However, there is no significant difference in relative levels of GAD mRNA in whole brains at e16.5 between control and Hes1 null animals (Supplementary Fig. 1). These data indicate that disruption in selective GABA signaling surrounding the SON and PVN may affect the boundary of AVP neuron expression within this region.

To establish the AVP-positive cell placement within the PVN and SON once cell migration and nuclei formation is complete, we compared these regions in Hes1 null and control animals at e18.5. There is continued misplacement of AVP-positive cell bodies within the PVN and SON of Hes1 null embryos (Fig. 3). AVP-positive cells within the PVN are located in a distinct pattern on either side of the third ventricle in control animals (Fig. 3A), but are more diffusely expressed in Hes1 null mice (Fig. 3B). Additionally, while the SON of control animals consists of a tight cluster of AVP-positive cell bodies superior to the optic nerve (Fig. 3C), Hes1 null mice show AVP-positive cells following the border of the mesenchyme (Fig. 3D), consistent with the scattered expression of medial AVP-positive cells found near the SON at e16.5 (Fig. 2D). There is no significant difference in the total number of AVP positive cells in the mid PVN and SON between control (PVN, 30.6±0.8; SON, 29.8±0.5) and Hes1 null animals (PVN, 31.2±0.5; SON, 30.7±0.3) at e18.5.

Fig. 3. Loss of Hes1 causes disruption of AVP cell body placement in the PVN and SON at e18.5.

Fig. 3

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Immunodetection of arginine vasopressin (AVP) cell bodies in coronal sections (red, co-labeled with nuclei stained with DAPI in blue) shows robust staining within the paraventricular nucleus (PVN; A, dotted box). In contrast, AVP-positive cells in Hes1 null animals extend outside this region (B, dotted box). AVP-positive neurons within the superior optic nucleus (SON) form a tight cluster in control embryos (C), while they sit in a linear and more diffuse pattern in the SON of Hes1 null animals (D). Forty sections from 4 individual embryos were examined for each genotype. Scale bar indicates 50μm.

Hes1 is necessary for formation of the PL

Given the alterations in placement of AVP-positive cells within the SON and PVN, we examined whether the axons of these neurons reach their target, the PL. AVP axons originating from the PVN project laterally to the SON and then return to the center of the head to travel through the median eminence (ME) and reach the PL at e18.5. Using immunohistochemistry techniques to analyze e18.5 pituitaries, we found that control animals show robust expression of AVP-positive terminals within the PL (Fig. 4A). In contrast, Hes1 null mice show a decrease in AVP-positive axon terminals within the hypomorphic PL, indicating some but not all AVP-positive axons reach their target in these mice (Fig. 4B). We then examined whether the AVP axons of Hes1 null mice reach their first target, the ME, before they terminate within the PL. We found robust AVP expression within the ME of control animals (Fig. 4C). However, Hes1 null animals have a decrease in AVP axons within the ME and AVP-positive axons project laterally in the same plane of section, anterior to the PL (Fig. 4D). The aberrant cluster of AVP-positive projections could account for the reduction of AVP-positive terminal axons within the PL in Hes1 null mice, as axons appear to be projecting elsewhere, lateral to the ME.

Fig. 4. Loss of Hes1 results in pituitary hypoplasia, reduction of AVP neurons in the posterior lobe, and AVP axon misguidance at e18.5.

Fig. 4

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Immunodetection of arginine vasopressin (AVP) axon terminals in coronal sections (red, nuclei stained with DAPI in blue) fill the posterior lobe (PL) of control animals (A), while AVP-positive axons are substantially reduced in the PL of Hes1 null animals (B). AVP-positive axons are present in the median eminence (ME) of Hes1+/+ mice (C), but are reduced in Hes1 null animals and are present in an ectopic cluster lateral to the ME (D). Thirty sections from 4 individual embryos were examined for each genotype. Scale bar indicates 50μm.

Altered axonal trajectory of AVP neurons in Hes1 null mice

In order to further visualize the axons travelling from the ME to the PL at e18.5 in Hes1 null mice, we analyzed sections from heads embedded sagittally 30° from the horizontal plane (Fig. 5). In control animals, AVP-positive cells within the SON are visualized at the level of the AL (Fig. 5A). In contrast, the same level of the AL within the Hes1 null embryos contains AVP axons traveling through the ME as well as AVP-positive cells within the SON (Fig. 5B), indicating that the ME is misplaced in the head of Hes1 null animals. In a more posterior section, both the AL and IL are visualized in control animals, as well as a cluster of AVP-positive cell bodies, likely the SON (Fig. 5C). At the same level containing the AL and IL, Hes1 null animals also display AVP-positive axons traveling through the ME and AVP-positive cell bodies and axon terminals in a lateral cluster (Fig. 5D). Posteriorly, the AVP axons traveling through the ME to reach the PL can be visualized in control animals (Fig. 5E). However, axons traveling through the ME are located more anteriorly in Hes1 null animals, in a section containing the AL and IL. Therefore, in a more posterior section containing the PL, Hes1 null animals display no ME or traveling AVP axons, and a cluster of AVP-positive cells and axon terminals located in the same region as the SON in more anterior sections (Fig. 5F, 5F′). These findings could explain the reduction in the amount of AVP-positive axon terminals within the PL of Hes1 null animals, as AVP axons traveling through the ME are apparent far more anteriorly than control animals and are likely misguided as a result. Regardless, Hes1 is clearly necessary for proper axon migration of AVP neurons to their targets, the ME and the PL. These findings, compiled with data from AVP cell body placement, are summarized in Fig. 6.

Fig. 5. Loss of Hes1 causes misplacement of the median eminence and ectopic AVP axon termination at e18.5.

Fig. 5

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Parasagittal sections cut 30° to the horizontal plane (A, inset), show immunodetection of AVP neurons (yellow, nuclei stained in red) in a cluster superior to the optic nerve in the supraoptic nucleus (SON) at the level of the anterior lobe of the pituitary (AL; A, dotted box). At the same level, Hes1 null animals show a smaller AL, AVP axons traveling through the median eminence (ME), as well a cluster of AVP neurons lateral to the ME (B, dotted box). More posteriorly, AVP neurons are still present in the SON of control animals (C, dotted box). In Hes1 null animals, AVP-positive neurons are present superior to the optic nerve in the SON (D, dotted box) and AVP axons can be detected traveling through the ME. At the level of the posterior lobe (PL) in control animals, AVP-positive axons are visualized traveling through the ME to their target, the PL (E). However, Hes1 null animals show only clusters of AVP axons at this level (F, dotted box), as well as AVP axons in a smaller PL. F′. Magnification of the axon processes in F. Thirty sections from 3 individual embryos were examined for each genotype. Scale bar indicates 50μm.

Fig. 6. Schematic representation of changes in AVP cell body placement and AVP axonal trajectory in Hes1 null mice.

Fig. 6

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Control animals show arginine vasopressin (AVP) positive cell bodies (blue circles) within the region of the paraventricular nucleus (PVN) and supraoptic nucleus (SON) and few AVP immunopositive cell bodies at the level of the median eminence dorsally (ME; A). AVP axons (pink lines) travel from AVP cell bodies in the PVN to the SON and project medially though the ME to reach the posterior lobe (PL; A). Hes1 null animals display AVP-positive cell bodies outside of the PVN and SON, as well as AVP positive cell bodies at the level of the ME and PL in a more ventral region compared to controls (B). AVP axons travel from the PVN to the SON and are found ectopically at the level of the ME, which itself is aberrantly located closer to the PL in Hes1 null mice. Fewer AVP axons terminate in the PL of Hes1 null animals, which is frequently smaller compared to controls (B).

Hes1 affects peptide content within the pituitary

Given the abnormal projection of AVP axons and the reduction of AVP expression within the PL, we hypothesized that Hes1 null pituitaries may have altered peptide content compared to controls. Previous studies have shown that Hes1 is important for proper differentiation of peptide- producing cell types within the AL and IL; specifically, Hes1 null mice display a loss of pro-opiomelanocortin (POMC) cells within the IL, and a reduction in proteins produced from POMC cleavage, such as melanocyte-stimulating hormone (αMSH) (Raetzman, Cai, Camper. 2007). Reduction in αMSH levels is coincident with reduced prohormone convertase 2 (PC2) expression in the IL of Hes1 null mice, an enzyme necessary to cleave POMC into MSH (Raetzman, Cai, Camper. 2007). We used MALDI-MS to analyze peptide content in Hes1 null and control pituitaries at e18.5. Interestingly, POMC [108–120], [103–120], [141–162] and αMSH were detected only in control animals (Fig. 7, Table 1). The fact that POMC and αMSH were not detected in Hes1 null pituitaries correlates with previous results reporting differences in melanotrope specification within Hes1 null pituitaries, and the reduction in PC2 within the intermediate lobe of Hes1 null animals at e18.5 (Raetzman, Cai, Camper. 2007).

Fig. 7. Loss of Hes1 affects peptide content within the pituitary at e18.5.

Fig. 7

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MALDI MS profile comparing relative intensities of peptides by mass/charge (m/z) detected in Hes1 null and control pituitaries using an acidified acetone extraction method. Somatostatin [92–100] and Pro-AVP [151–168] were found only in Hes1 null pituitaries. Control animals showed various forms of pro-opiomelanocortin (POMC) as well as detection of melanocyte stimulating hormone (MSH) not found in Hes1 null pituitaries. Both Hes1 null and control pituitaries contained cholecystokinin (CCK-8) and ProAVP/AVP.

Table 1.

Summary of peptides identified by MALDI-MS in Hes1 null and control animals at e18.5.

Prohormone/Peptide Sequence m/z Hes1+/+ Hes1−/−
Unknown 858.89 X X
Somatostatin [92–100] SNPAMAPRE 972.82 X
ProAVP/AVP C*YFQNC*PRGa 1084.90 X X
CCK-8 DY(PO4)MGWMDFa 1142.93 X X
ProAVP [151–168] VQLAGTRESVDSAKPRVY 1976.85 X
Unknown 1330.19 X
POMC [108–129] VWGDGSPEPSPREa 1411.59 X
MSHα [124–136] SYSMEHFRWGKPVa 1622.65 X
POMC [103–120] AEEEAVWGDGSPEPSPREa 1941.71 X
POMC [205–222] (Ac)YGGFMTSEKSQTPLVTLF 2047.84 X
POMC [141–162] RPVKVYPNVAENESAEAFPLEF 2505.78 X
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Additionally, ProAVP/AVP-related peptides were detected in both Hes1 null and littermate controls, and a C-terminus form of ProAVP [151–168] was found only in Hes1 null animals (Fig. 7, Table 1), which may be related to levels of detection or physiological differences in processing. Surprisingly, a form of SS (m/z 972.82) was detected in Hes1 null animals only (Fig. 7, Table 1). Although SS is released into the ME to act on the AL through the portal vasculature, it should not be detectable in the pituitary extracts at this age. Overall, these data suggest that Notch signaling, directly or indirectly, may impact peptide processing, as Hes1 null pituitaries display a different peptidomic profile compared to controls.

Hes1 loss leads to reduction of SS neurons in the aPV, and aberrant axonal tracts to and expression within the PL

Based on the MS observations of alterations in SS peptide content within the pituitary of Hes1 null animals at e18.5, we investigated SS protein expression in these animals using immunohistochemistry. In the aPV of control animals, SS-positive neurons are found lateral to the third ventricle (Fig. 8A), and the numbers of SS-positive neurons appear to be reduced in Hes1 null animals (Fig. 8B). Additionally, we found alterations in SS-positive axon projections, which normally project from the aPV to the median eminence. At e18.5, control animals show no expression of SS-positive axons lateral to the ME (Fig. 8C), while Hes1 null mice display abnormal SS-positive axonal tracts in this region (Fig. 8D). Abnormal SS-positive tracts were also observed along the mesenchyme border immediately dorsal to the PL at e18.5, and were not localized to mesenchymal or blood cells (data not shown). Additionally, SS cell bodies were aberrantly found in the PL of Hes1 null pituitaries (1.75 cells/pituitary ±0.25, n=4, Fig. 8F), compared to WT mice (0 cells/pituitary n=4; Fig. 8E). These alterations in SS axon pathfinding and termination, together with the AVP data described, indicate that Hes1 may play a role in the guidance of hypothalamic axons to the pituitary.

Fig. 8. Loss of Hes1 results in reduced SS-positive cells in the aPV, altered SS tracts and aberrant expression of SS in the posterior lobe at e18.5.

Fig. 8

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Immunodetection of somatostatin (SS) cell bodies (red, co-labeled with nuclei stained with DAPI in blue) in the mid-aPV nucleus shows SS-positive cells surrounding the third ventricle (3V) in control animals (A), while Hes1 null animals show a decrease in SS-positive cells in this region (B). At the level of the medial median eminence (ME), there are no SS-positive tracts lateral to the ME in control animals (C), while Hes1 null animals show SS-positive axons along the ventral border of the brain (D). Within the posterior lobe, control animals display no SS-positive cells in the posterior lobe (PL; 0 cells, n=4) or intermediate lobe (IL; E). However, Hes1 null pituitaries show aberrant expression of SS-positive cells in the PL (arrow; 1.75 cells ±0.25, n=4) and immunoreactivity in the IL (F). Forty sections from 4 individual embryos were examined for each genotype. Scale bar indicates 50μm.

DISCUSSION

The principal role of Notch signaling during cortical development is to maintain neural progenitor identity and suppress neuronal differentiation (Schuurmans, Guillemot. 2002). Loss of the Notch effectors Hes1 and Hes3 causes premature neuron formation in the mid/hind brain and subsequent loss of mid/hind brain structures (Hirata, et al. 2001). Within the developing endocrine pituitary AL, loss of Hes1 causes a reduction in cell proliferation and decreased numbers of hormone-producing cells (Raetzman, Cai, Camper. 2007). Based on this data and the reduction in size of the PL within Hes1 null animals, we would have expected that Hes1 null mice would display premature differentiation of neuronal precursors, leading to a reduction in VD neuronal number. Consequently, we would have also expected decreased numbers of differentiated, AVP-positive neurons within the hypothalamic PVN/SON regions. Instead our results indicate that Hes1 alone does not affect proliferation within the developing VD, or formation and specification of AVP neurons within the PVN and SON. It is likely that other factors in the Notch signaling pathway, expressed in the same regions as Hes1, such as Hey1 (Raetzman, et al. 2006) or Hes5 (Kita, et al. 2007), may be compensating for the loss of Hes1 in these animals. It is also possible that earlier effects on proliferation and cell death escaped our detection, as our studies began analysis at e10.5, when the primordial posterior pituitary is already reduced in size.

Given the reduced size of the PL found in Hes1 null mutants, our studies focused on the specification, placement and projection of AVP neurons, which terminate in the PL. Our studies indicate that Hes1 is not necessary to form the appropriate number of AVP neurons. However, we found that the number of SS-positive neurons within the aPV appears reduced in Hes1 null mice. These data indicate that Hes1 may play a distinct role in the differentiation or placement of SS neurons in the aPV. Importantly, SS neurons require expression of Sim2 for proper differentiation, while AVP neurons do not (Goshu, et al. 2004). Within the developing spinal cord, the Notch signaling receptor Notch1 regulates the specification of certain lineages of interneurons. Loss of Notch1 results in overproduction of V2 neurons at the expense of motor neuron formation due to specific regulation of Lhx3+ cells (Yang, et al. 2006). Therefore, it is possible that Hes1 may regulate transcription factors required for specific cell lineages and not others, allowing for the precision and specificity of normal neuronal differentiation. Additionally, Notch signaling may be acting through Hes1 in a specific temporal context, such that differentiation signaling for AVP neurons acts at a different developmental time compared to differentiation of SS neurons, affecting SS but not AVP neuron number. Though not addressed in our study, it is possible that loss of Hes1 affects the formation and placement of OT neurons, which also terminate in the PL and originate from the PVN and SON.

Once AVP and SS neurons are born and differentiated, they migrate to the appropriate hypothalamic nuclei to exert their function. Our results indicate a novel role for Hes1 in migration of AVP cell bodies within the PVN and SON. Although Hes1 may be necessary intrinsically in AVP neurons to allow them to migrate appropriately, it is also likely that loss of Hes1 results in changes in the surrounding environment, which subsequently affects AVP cell body placement. For instance, we found that GABAergic neurons were randomly distributed within the SON and PVN in Hes1 null mice compared to controls. It is possible that Hes1 is required to limit the extent of GABAergic differentiation in the developing HP because blocking Hes1 in human neural stem cells initiates GABAergic differentiation through induction of cyclin-dependent kinase inhibitor p21 (Kabos, Kabosova, Neuman. 2002). GABAergic neurons have been shown to surround the PVN during development in order to establish the boundaries of AVP neurons within this region (McClellan, Stratton, Tobet. 2010). It is possible that aberrant expression of GABAergic neurons within the developing PVN and SON affects delineation of PVN and SON boundaries, and contributes to the misplacement of AVP neurons in Hes1 null mice.

In normal animals, once AVP neurons reach their nuclei, they begin to extend their axons to their targets. Specifically, AVP neurons in the PVN travel ventrally and laterally to the SON and then all axons project back to the midline for entry into the ME and PL. Previous studies have implicated Notch signaling in axonal targeting within the cerebral cortex and hippocampus in conjunction with the extracellular matrix molecule Reelin (Alcantara, et al. 1998; Sibbe, et al. 2009). Reelin-deficient mice have reduced levels of cleaved NICD, and loss of Notch signaling in developing neurons results in faulty migration. Furthermore, overexpression of NICD mitigates cortical neuronal migration defects associated with Reelin null mice (Hashimoto-Torii, et al. 2008). Reelin mRNA is also expressed in the primordial PVN, SON and aPV during embryonic development (Alcantara, et al. 1998), although its relationship with members of the Notch signaling pathway, and Hes1 in particular, during hypothalamic development is unclear. It is possible that Hes1 may interact with Reelin within the developing endocrine hypothalamus to guide axons to their targets, the ME and pituitary.

In our study, the global loss of Hes1 may affect the developmental environment, such that normal guidance cues are dysregulated and axons are unable to navigate normally. In Hes1 null animals, the ME is clearly misplaced within the head, which may contribute to the aberrant cluster of AVP axons and the reduced number of AVP axons within the PL. The trophic components in and around ME during embryonic development are not well characterized, but support cells within the ME, called tanycytes, modulate hypothalamic neurons that travel through it in the mature HP (Givalois, et al. 2004; Prevot, et al. 2007; Prevot, et al. 2010). There is some evidence that hypothalamic gonadotropin-releasing hormone (GnRH) axons use cues from their target, the ME, for successful navigation (Rogers, Silverman, Gibson. 1997), and tanycytes may mediate these cues. Hes1 null animals display premature differentiation and eventual depletion of radial glial cells (Hatakeyama, et al. 2004), which establish a structural framework to facilitate and direct axonal migration (Metin, et al. 2008). It is possible that loss of Hes1 affects the proper development of support cells such as tanycytes in the ME, contributing to dysregulated axon guidance. Additionally, loss of Hes1 may affect proper formation of pituicytes, the glial cell population within the PL, which could contribute to the misguided axon and hypoplastic pituitary phenotype.

Perhaps the most vital contributions to the identity of a hypothalamic neuron are the peptides that it synthesizes and releases. Our data have uncovered a novel role for Notch signaling in the peptide content of hypothalamic neurons. Peptide processing occurs within the cell body of hypothalamic neurons, with peptides transported through the axons and released through axon terminals in the ME or PL. Our results indicate that Hes1 may play a role in peptide processing, as Hes1 null pituitaries display different peptidomic profiles compared to controls, including alterations in MSH, AVP and SS peptide content.

There is precedence for other signaling pathways crucial for early embryonic development affecting neuropeptide processing. Homozygous Fgf8 hypomorphic mice show deficiency in post-translational processing and cleavage of the OT prohormone found in OT neurons of the PVN and SON (Brooks, Chung, Tsai. 2010). Additionally, Fgf2 treatment has been shown to cause incomplete processing of GnRH neurons such that the prohormone is cleaved into intermediate peptide products extended at the C-terminus (Wetsel, Hill, Ojeda. 1996). Taken together with our data, it is possible that Hes1 may play a role not only in the migration and projection of hypothalamic neurons, but also in their functional delivery of neuropeptide to the pituitary.

A specific and well-regulated cellular environment is crucial during development in order to establish proper connections between the hypothalamus and pituitary. Early in embryonic development the primordial hypothalamus and pituitary are in physical contact and require reciprocal signaling. Given that Hes1 is required for proper formation of the pituitary (Raetzman, Cai, Camper. 2007), it is possible that Notch signaling is required when contacting oral ectoderm and neural ectoderm are actively differentiating. Specifically, guidance signals from the oral ectoderm, fated to become pituitary, may be necessary to guide hypothalamic axons later in development. Notch signaling could also be a necessary intrinsic signal within neural ectoderm, and acts to guide AVP and SS hypothalamic neurons.

Notch signaling within the developing oral and neural ectoderm could also affect other signaling cascades, such as the FGF and BMP pathways. Although previous studies have shown that loss of Hes1 does not affect Fgf10 mRNA expression (Raetzman, Cai, Camper. 2007), it is possible there are changes in levels of FGF protein. Additionally, other morphogens released from the developing diencephalon and infundibulum, such as BMPs, may affect early PL formation. Future studies utilizing tissue-specific Notch knockout mice will clarify whether autonomous Notch signaling is required in the developing hypothalamus, pituitary, or both, for proper development and migration of hypothalamic neurons and their axons.

Supplementary Material

01. Fig. S1. Relative levels of AVP and GAD mRNA are not changed in Hes1 null brains at e16.5.

qRT-PCR reveals that there is no change in the level of arginine vasopressin (AVP) (control 1.1±0; null 1.2±0.1) and glutamate decarboxylase (GAD) (control 1.0±0.1; null 1.0±0.1) mRNA between control and Hes1 null whole brains at e16.5 (n=5).

02

Acknowledgments

Grant Sponsor: NIH Grant R01 DK076647, NIH Grant T32 HD007333 and NIDA Grant P30DA018310

This work was supported by NIH Grant R01 DK076647, NIH Grant T32 HD007333 and NIDA Grant P30DA018310. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIDA or the National Institutes of Health. We would like to thank Katherine Brannick for her assistance with the mouse colony and Gloria Hoffman for her technical advice.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Fig. S1. Relative levels of AVP and GAD mRNA are not changed in Hes1 null brains at e16.5.

qRT-PCR reveals that there is no change in the level of arginine vasopressin (AVP) (control 1.1±0; null 1.2±0.1) and glutamate decarboxylase (GAD) (control 1.0±0.1; null 1.0±0.1) mRNA between control and Hes1 null whole brains at e16.5 (n=5).

NIHMS283319-supplement-01.doc (34.5KB, doc)
02
NIHMS283319-supplement-02.pdf (85.1KB, pdf)

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