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National Institutes of Health, National Institute of Neurological Disorders and Stroke, Cellular and Developmental Neurobiology Section, Bethesda, Maryland 20892-4156
Address all correspondence and requests for reprints to: Susan Wray, National Institutes of Health, National Institute of Neurological Disorders and Stroke, Cellular and Developmental Neurobiology Section, 36 Convent Drive, Bethesda, Maryland 20892-4156. E-mail: wrays{at}ninds.nih.gov.
Abstract
GnRH-1 is a decapeptide hormone that regulates gonadal maturation and fertility. In brain, GnRH-1 is secreted by neurons residing mainly in the preoptic/hypothalamic area. These neurons arise from cells in the nasal placode during embryonic development. GnRH-1 mRNA and peptide in the nonhypothalamic region have been described, suggesting other functions of GnRH-1. This paper describes for the first time the expression of GnRH-1 in developing incisors in mice. At embryonic day (E) 12.5, GnRH-1 mRNA and peptide were localized in cells in oral and dental epithelia. At postnatal day (P) 6, before incisor eruption, GnRH-1 was expressed in cells in dental epithelial-derived structures that include the papillary layer, outer dental epithelium, stellate reticulum, stratum intermedium, and enamel-secreting ameloblast cell layer. GnRH-1 expression correlated with cell maturity, becoming stronger in cells farther away from the proliferative zone. From E12.5 through P6, GnRH-1 expression was not detected in neural crest-derived dental mesenchyme or in mesenchyme-derived structures that include dental papilla, dental follicle, and dentin-secreting odontoblast. In addition, GnRH-1 expression was not detected in molars, indicating that expression of GnRH-1 is differentially regulated in incisor vs. molars, with only the former exhibiting continuous growth in this species. In homozygous hypogonadal mice at P1, GnRH-1 peptide expression was not detected, yet incisors were present. However, morphological changes in cells between dental follicle and ameloblast cell layer were noted. Taken together, our results indicate that GnRH-1 expression, although not essential for initiation and formation of incisors, may be important in maturation and/or maintenance of these placodally derived structures.
GnRH-1 IS A decapeptide hormone that is important for maturation of gonads and maintenance of reproduction in vertebrates (1, 2, 3). The importance of GnRH-1 in reproduction is exemplified in hypogonadal (hpg) mice, which have a deletion mutation in the GnRH-1 gene (4). In these mice, the testis and ovary fail to mature postnatally and are consequently infertile (5). Rescue of hypogonadism and infertility was achieved with GnRH-1 gene therapy (4, 6), or transplantation of hypothalamic grafts (7) or of GnRH-expressing cells (8) into the hypothalami of hpg mice.
GnRH-1 neurons arise from the nasal region and migrate, in association with olfactory axons, into the hypothalamus during development (9, 10). Failure of GnRH-1 neurons and olfactory axons to enter the central nervous system results in loss of the sense of smell as well as reproductive dysfunction, such as that seen in patients with Kallmann’s syndrome (11). However, GnRH-1 mRNA and/or peptide has been localized in other brain regions (tectum, lateral septum, bed nucleus of stria terminalis, and amygdala) and other tissues including splenocytes, lymphocytes, liver, heart, skeletal muscle, kidney, placenta, breast, pancreas, and gonads (12, 13, 14, 15, 16, 17). The expression of GnRH-1 in these tissues suggests additional novel functions of GnRH-1 (17).
This paper investigates GnRH-1 expression during tooth development. The development of the tooth is a dynamic process that involves tooth morphogenesis and cytodifferentiation (18). This process involves the interaction of ectoderm-derived dental epithelial cells and underlying neural crest-derived mesenchymal cells, an event that is similar to the development of tissues such as hair, scales, limbs, and in particular craniofacial invagination of the olfactory and respiratory epithelia (19, 20). This report documents differential expression of GnRH-1 in teeth, being present in incisors and not in molars. GnRH-1 is detected early in incisor development and is maintained postnatally.
Materials and Methods
Mice
NIH Swiss Webster mice (Division of Cancer Treatment, Frederick, MD) at different stages of development [embryonic day (E)11.5 through adult] and JAX GEMM HPG/BmJ mice at postnatal day (P)1 were used. Homozygous hpg mice were generated in-house by mating heterozygous hpg mice obtained from The Jackson Laboratories (Bar Harbor, ME). Wild-type, heterozygous, and homozygous hpg screening was done by PCR using primers 5'-TATGGCTTACAGTTCCAGCG-3' (forward) and 5'-AGGCTTGGAGAGCTGTAAGG-3' (reverse 1), which are directed to the intronic region of the GnRH-1 gene between exon II and exon III, and a primer 5'-GTTTCAGTGCATCCTCTCAGG-3' (reverse 2), which binds downstream of the 33.5-kb region of the GnRH-1 gene that is deleted in homozygous hpg mice. Adult mice were killed in a carbon dioxide chamber, followed by decapitation. Embryos were removed from timed pregnant females, and fresh, frozen sections (12–16 µm) of head or whole embryo were cut on a cryostat (model CM3050 S; Leica, Deerfield, IL). P1 and P5 mice were decapitated, immediately frozen, and stored (at –80 C) until sectioning (12–16 µm). Tissues from embryos and from P1, P5, and nonpregnant adult mice were obtained for RNA isolation. All procedures using animals were approved by the Animal Care and Use Committee of National Institute of Neurological Disorders and Stroke/National Institutes of Health (NINDS-NIH) and tissues were obtained in accordance with the NINDS-NIH guidelines.
In situ hybridization histochemistry
Mouse sections were hybridized as previously described (21) with deoxynucleotide probes (S-35 labeled at 3'end). Two antisense probes (5 pmol) were used: a 48-mer (5'-TTCAGTGTTTCTCTTTCCCCCA-GGGCGCAACCCATAGGACCAGTGCTG-3') or a 36-mer (5'-CTTCCGACGAGGT-CGGTCGTGACCAGGATACCCAAC-3'). One sense probe complementary to the 36-mer described above was also used (negative control). Briefly, sections (16 µm) were fixed (in 4% formaldehyde), permeabilized (in 0.3% Triton X-100/0.05 M EDTA/0.1 M Tris, pH 8), acetylated (in 0.25% acetic anhydride/0.1 M triethanolamine HCl), rinsed (in 2x saline sodium citrate), dehydrated and delipidated, rinsed with ethanol, and air-dried. Sections were hybridized in a humidified chamber (37 C, 
16 h). After hybridization, slides underwent low- and high-stringency washes followed by dehydration. After exposure to x-ray film (3–7 d), slides were dipped in emulsion (NTB3; Eastman Kodak, Rochester, NY) and exposed for 3–4 wk. Emulsion covered slides were developed, counterstained with methyl green, and coverslipped.
Immunocytochemistry
Three GnRH-1 antibodies were used to ensure specificity of the signal: SW1 (1:3000; Ref.22), LR5 (1:15000, generous gift of Drs. Benoit and Guilleman, The Salk Institute, San Diego, CA), and LHRH-A (1:1500; ImmunoStar, Hudson, WI). A similar GnRH-1 staining pattern was obtained with all antibodies. Staining was performed using standard avidin/biotin complex-horseradish peroxidase procedures (22). Briefly, fresh, frozen embryonic and brain sections were fixed (in 4% formaldehyde for 1 h). Pretreatment of sections with 3% hydrogen peroxide in methanol (5 min) was performed to eliminate endogenous peroxidases. Sections were then blocked (in 10% normal goat serum/0.3% Triton X-100/0.1% sodium azide for 1 h), and incubated in primary antibodies or 10% normal goat serum overnight (4 C). Slides were washed (in PBS) and incubated in biotinylated goat antirabbit (1:500; Vector Laboratories, Inc., Burlingame, CA) followed by avidin/biotin complex. Chromagens used were diaminobenzidine (Sigma-Aldrich, Co., St. Louis, MO) or Vector SG (Vector Laboratories, Inc.).
RT-PCR
Mouse tissues (hypothalamus, tails, limbs, nose with upper jaw, and lower jaw) from E11.5 to adult stages were obtained. Total RNA extraction was conducted using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) following the manufacturer’s protocol. Briefly, 1.0 ml RNA STAT-60 for 50–100 mg tissue was used during homogenization. Chloroform (0.2 ml/ml homogenate) was used in the RNA extraction and isopropanol (0.5 ml/ml solution) was added to the aqueous layer to precipitate RNA. RNA pellet was washed with 75% ethanol, air-dried, and resuspended in diethyl pyrocarbonate-treated water.
One nanogram of RNA was used for the RT-PCR using the AccessQuick RT-PCR System (Promega, Madison, WI). The reaction was performed at 48 C for 45 min; 94 C for 2 min; 40 cycles of 94 C for 30 sec, 55–65 C for 1 min, and 68 C for 2 min; and 68 C for 7 min. A second round of PCR was conducted using 1 µl of the RT-PCR product and performed at 94 C for 5 min; 40 cycles of 94 C for 30 sec, 55–65 C for 30 sec, and 72 C for 2 min; and 72 C for 10 min. The PCR products were resolved on a 1.5% agarose gel by electrophoresis.
Primers that bind to nucleotides encoded from exons I and IV were used to evaluate for full-length GnRH-1 transcript: F1 (5'-GCTAGGCAGACAGAAACTTCG-3') and R2 (5'-GGTGTTGTGGATCCACCTGG-3') in RT-PCR and F1 and nested R3 (5'-GCATCTACATCTTCTTCTGCC-3') in the second PCR. The presence of the truncated transcript of GnRH-1 in hpg mice was confirmed using primers that binds to nucleotides encoded from exons I and II: F1 and 5'-TGGAAAGACTCAACCAAGTGTT-3' in RT-PCR and F1 and nested 5'-CAGTGTTTCTCTTTCCCCCAG-3' in the second PCR. Amplification of glyceraldehyde-3-phosphate dehydrogenase with primers 5'-GTCATCATCTCCGCCCCTTC-3' and 5'-ATCCAGGGACACATTGG-3' was used to determine the integrity of RNA sample and in normalization of PCR products.
Results and Discussion
Tooth development involves a dynamic interaction between oral epithelium and underlying mesenchyme (18). The odontogenic placode (epithelial thickening) formed at E9/10 marks the beginning of tooth morphogenesis (23). This is followed by formation of dental lamina at E11.5 (Fig. 1A-1
), evagination of dental epithelium and condensation of mesenchyme at E12.5–E13.5 (bud stage; Fig. 1A-2), cell differentiation and morphogenesis at E14.5 (cap stage; Fig. 1A-3) and E15.5 (early bell stage; Fig. 1A-4), and by late bell stage (
E16.5) formation of dentin and enamel occurs, and their corresponding mineralization follows (23, 24, 25, 26, 27, 28).
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, C–F). At E14.5–P6, GnRH-1 expression was detected in cells associated with the epithelial-derived structures of incisors: inner and outer dental epithelia, stellate reticulum, stratum intermedium, and ameloblast layer. Figure 2A shows GnRH-1 peptide expression at P5 (late bell stage) in cells on the labial side of the incisors extending about two thirds of its length, from the more incisal (distal, toward the tip) to the more proximal region. In the more incisal region, robust expression of GnRH-1 peptide was localized in cells in the papillary cell layer (Fig. 2B'). This layer is a condensed structure composed of outer dental epithelium, stratum intermedium, and stellate reticulum. Weaker immunostaining was present in the more proximal part of the growing incisor (Fig. 2A'). GnRH-1-positive staining in this region was localized in cells in the outer dental epithelium, stratum intermedium and stellate reticulum, and enamel-secreting ameloblast cells. GnRH-1 immunostaining was absent at the tip of the incisors where the cells in the ameloblast and papillary layers are no longer present and in the most proximal (apical) aspect of the developing incisors where the germinal zone (mitotic cells that add to the growing incisor) lies (Fig. 2A). A similar pattern of staining is detected in the lower incisors (Fig 2, D and E). These results suggest that GnRH-1 expression is related to maturation of the epithelial-derived structure of the incisors, specifically that of cells comprising the ameloblast and papillary layers.
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). In addition, no GnRH-1 mRNA or peptide was detected in the developing molars (Fig. 2C), suggesting differential factors are present in molars and incisors.
After P6, histological sections become difficult to obtain because of bone formation; hence, mRNA expression analysis in later age was conducted by RT-PCR on isolated lower jaws. The GnRH-1 gene has four exons: exon I encodes for the 5' untranslated region; exon II encodes for signal peptide, GnRH-1 decapeptide, and part of GnRH-associated peptide; and exons III and IV encode for the rest of GnRH-associated peptide and 3' untranslated region (29). Using primers directed to exons I and IV of the GnRH-1 gene, a 320-bp RT-PCR product was present in the hypothalamus of an adult mouse, in nose with upper jaw at E11.5, and in lower jaw at E12.5 but was not detected in the lower jaw at E11.5 (Fig. 3, left panel). GnRH-1 mRNA and peptide are produced by GnRH-1 neurons that reside in the nasal placode/nasal pit at E10.5–E11.5 and migrate into the brain to reside in a diffuse location in the septohypothalamic region (22, 30). Thus, the same size RT-PCR product in E12.5 lower jaw, in E11.5 nose, and adult hypothalamus indicates that the GnRH-1 transcript in the incisors is a full-length transcript. Full-length GnRH-1 transcript was also detected in the lower jaws of weanling and adult mice (Fig. 3, right panel), suggesting continued expression of GnRH-1 in incisors of adult mice.
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, right panel). Homozygous hpg mice at P1 have incisors (Fig. 4), suggesting that GnRH-1 is not necessary for initiation and formation of these teeth. At this stage, a profound histological difference in the developing incisors of homozygous hpg vs. wild-type mice was detected. In the homozygous hpg mice, cells in the papillary layer appeared loosely packed and spread further into the dental follicle compared with wild-type mice (Fig. 4, D–F). This suggests that GnRH-1 expression in the incisor is associated with cell-cell interaction and/or cell-matrix adhesion. Although the enamel layer appears to be present in hpg mice, the effect of knockdown of GnRH-1 expression on mineralization of the enamel and on size and shape of mature incisor needs to be further evaluated.
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Footnotes
Abbreviations: E, Embryonic day; hpg, hypogonadal; P, postnatal day.
Received March 25, 2004.
Accepted for publication May 12, 2004.
References
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  K. Walters, I. N. Wegorzewska, Y.-P. Chin, M. G. Parikh, and T. J. Wu Luteinizing Hormone-Releasing Hormone I (LHRH-I) and Its Metabolite in Peripheral Tissues Experimental Biology and Medicine, February 1, 2008; 233(2): 123 - 130. [Abstract] [Full Text] [PDF]
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 J S. Mohamed and I A Khan Molecular cloning and differential expression of three GnRH mRNAs in discrete brain areas and lymphocytes in red drum. J. Endocrinol., March 1, 2006; 188(3): 407 - 416. [Abstract] [Full Text] [PDF]
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