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Endocrinology, doi:10.1210/en.2005-1620
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Endocrinology Vol. 147, No. 5 2237-2244
Copyright © 2006 by The Endocrine Society

Ablation of Persephin Receptor Glial Cell Line-Derived Neurotrophic Factor Family Receptor {alpha}4 Impairs Thyroid Calcitonin Production in Young Mice

Päivi H. Lindfors, Maria Lindahl, Jari Rossi, Mart Saarma and Matti S. Airaksinen

Neuroscience Center (P.H.L., J.R., M.S.A.), and Institute of Biotechnology (M.L., M.S.), University of Helsinki, FIN-00014 Helsinki, Finland

Address all correspondence and requests for reprints to: Matti S. Airaksinen, Neuroscience Center, Viikinkaari 4 (P.O. Box 56), University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: mairaksi{at}operoni.helsinki.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glial cell line-derived neurotrophic factor family receptor (GFR{alpha}) 4, the binding receptor for persephin, is coexpressed with the signaling Ret receptor tyrosine kinase predominantly in thyroid calcitonin-producing C cells. We show by in situ hybridization and immunohistochemistry that the functional, glycolipid-anchored form of GFR{alpha}4 is produced in mouse only in the C cells but not in parathyroid gland or in the brain. C cells expressed functional GFR{alpha}4 throughout postnatal development, whereas Ret expression in these cells decreased postnatally and was undetectable in adults. To understand the physiological role of GFR{alpha}4, we produced GFR{alpha}4-deficient [knockout (KO)] mice. No differences were observed between wild-type and GFR{alpha}4-KO littermate animals in growth, gross behavior, or viability. The number and morphology of the thyroid C cells were indistinguishable between the genotypes in both newborn and adult age. However, thyroid tissue calcitonin content was reduced by 60% in newborn and by 45% in 3-wk-old GFR{alpha}4-KO mice compared with wild-type controls. In contrast, thyroid calcitonin levels were similar in adult animals. Consistent with the reduced calcitonin levels, bone formation rate in juvenile GFR{alpha}4-KO mice was increased. In conclusion, this study indicates a novel role for endogenous GFR{alpha}4 signaling in regulating calcitonin production in thyroid C cells of young mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLIAL CELL LINE-derived neurotrophic factor (GDNF) family ligands [GDNF, neurturin, artemin, and persephin (PSPN)] signal through a receptor complex consisting of a glycosyl-phosphatidylinositol (GPI)-linked GDNF family receptor {alpha} (GFR{alpha}) subunit (GFR{alpha}1 to GFR{alpha}4) and the transmembrane tyrosine kinase Ret (for review see Refs.1 and 2). We and others (3, 4) have previously identified mammalian GFR{alpha}4 as the binding receptor for PSPN.

Exogenous PSPN can promote the survival of several types of embryonic neurons from the rat central nervous system in vitro but not of any peripheral neurons examined (5, 6). Moreover, PSPN is reported to protect mouse brain neurons from lesion-induced death in vivo (7, 8). However, it is not known whether these activities of PSPN are mediated via endogenous GFR{alpha}4. The physiological role of PSPN remains unclear because Pspn mRNA is expressed at very low levels in many tissues of embryonic and adult rodents, and PSPN-deficient mice show normal development and behavior (8).

Alternative splicing of the mouse Gfra4 gene produces GPI-anchored and transmembrane isoforms, as well as transcripts with premature stop codons (9). The transcript that encodes for the functional GPI-anchored form of GFR{alpha}4 with a functional signal sequence is predominantly expressed in endocrine cells of thyro-parathyroid gland, pituitary intermediate lobe, and developing adrenal medulla (9). Although abundant, most if not all Gfra4 transcripts in the mouse nervous system appear nonfunctional, lacking the signal sequence and having premature stop codons.

To assess the biological function of GFR{alpha}4 in vivo, we produced GFR{alpha}4 knockout (KO) mice by deleting most of the receptor gene. We focused our analysis on the thyroid C cells, the only cells known to coexpress functionally active GFR{alpha}4 and Ret in mammals (3, 9). We show, using specific antibodies, that GFR{alpha}4 protein is expressed only in the C cells, and coexpressed with Ret only in young mice. Our results indicate that GFR{alpha}4 is necessary not for the development of thyroid C cells, but for their function, namely for their transmitter production in young mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of GFR{alpha}4-KO mice
A mouse BAC clone 389B9 (Research Genetics, Huntsville, AL) that contains the Gfra4 locus (10) was digested with XbaI, and approximately 8-kb fragments were isolated and subcloned into a plasmid vector. They were screened by colony hybridization using a Gfra4 EST (AA823200; NCBI IMAGE consortium) as a probe. A 1440-bp KpnI-SacII fragment of the Gfra4 gene containing exons II–V was replaced with a 1.6-kb PGKneo cassette (Fig. 1AGo). This targeting vector was linearized with SfiI and electroporated into R1 embryonic stem cells. The cells were selected with geneticin and screened by Southern blot analysis (data not shown). Chimeras were produced by morula aggregation. The mutant Gfra4 allele was transferred into both C57BL/6JOlaHsd and 129SvHsd (Harlan UK Ltd., Bicester, UK) mouse backgrounds by backcrossing for at least five generations. Resulting offspring was genotyped by PCR (Fig. 1BGo) using primers 5'-CGA TTC GCA GCG CAT CGC CTT C-3', 5'-ATA CAA GCC TTT GAC AGC TTG C-3', and 5'-TGG ACA AGA TGC CTA CTG ACG-3' that give specific PCR products for wild-type (WT) (~450 bp) and mutant alleles (~800 bp). In most experiments, we used sex-matched (C57BL/6 x 129Sv) F1 hybrid WT and GFR{alpha}4-KO littermates obtained from intercrosses of the congenic heterozygous parents. Additional age- and sex-matched WT and GFR{alpha}4-KO mice were obtained from F1 homozygous matings (offspring of different parents to minimize the risk of genetic bias) and used in the calcitonin assays. All mice were given a standard chow diet (Teklad, 1% calcium). All animal experiments were approved by the Animal Research Ethics Committee at the University of Helsinki.


Figure 1
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  		FIG. 1. A, Targeting strategy. WT Gfra4 gene locus, targeting construct, and the targeted allele, in which exons 2–5 are replaced with a neo cassette. Arrowheads mark the PCR primer locations. B, Example of PCR genotyping. C, RT-PCR analysis shows strong expression of Gfra4 mRNA in WT and its absence in GFR{alpha}4 KO mouse thyroid. The lines contain similar levels of control Gapdh transcript.

 
RT-PCR
Total RNA was isolated from mouse tissues using RNAwiz reagent (Ambion, Austin, TX). RT reactions were performed using SuperscriptIII (Life Technologies, Rockville, MD). Full-length Gfra4 was amplified using the GC-rich PCR kit (Roche Applied Science, Basel, Switzerland) and primers P1 and P6 described in Ref.9 that produce 780- and 879-bp PCR fragments corresponding to the GPI-anchored and transmembrane forms of GFR{alpha}4, respectively. PCR was run 40 cycles using annealing temperature of 55 C. The amount of cDNA in samples was shown by amplification of a 449-bp Gapdh (glyceraldehyde-3-phosphate dehydrogenase) fragment using primers: 5'-GTG GAA GGG CTC ATG ACC ACA G-3' and 5'-GGA GTT GCT GTT GAA GTC GCA GG-3'.

In situ hybridization
In situ hybridization on cryosections was performed using probes for Ret and Gfra1 to Gfra4 mRNAs as described (9, 11). A probe specific for Gfra4 exon-1a was generated by PCR from the thyroid gland using the forward primer 5'-GTG CCT GCT GCC TTC CAG-3' and reverse primer 5'-AGA CCC CAG CAG CAA CAA CAG-3'. The resulting 127-bp PCR fragment contains part of the 5'UTR, exon-1a coding for the signal sequence and 5 bp downstream inside exon-2. The PCR fragment was cloned into pCRII vector (Invitrogen, Carlsbad, CA) and was verified by sequencing. The Gfra4 3' probe that contains 850 bp of the 3' end of Gfra4 including the 3' part of exon-4, exon-5, exon-6 and 3'UTR has been described before (9). Control sections hybridized with sense probe did not show labeling above background (data not shown).

Immunohistochemistry
Newborn, 3-wk-old and adult GFR{alpha}4-KO and WT littermate mice were anesthetized with an overdose of chloral hydrate (ip) and perfused transcardially with PBS followed by 4% paraformaldehyde. The thyroid glands with attached trachea and esophagus and other tissues (adrenal gland, brain, pituitary, and testis) were dissected, post fixed overnight, and immersed in 30% sucrose. Cryosections of the thyroid gland, serially sectioned at 15 µm, were immunostained with primary antibody against calcitonin (Santa Cruz, Santa Cruz, CA) and detected using ABC Elite kit (Vector, Burlingame, CA). Hoechst33258 (Molecular Probes, Eugene, OR) was included in embedding medium to stain the cell nuclei. All immunopositive profiles with clear nucleus were counted in every other section through the thyroid glands. For colocalization studies, 20-µm thyroid cryosections were double-stained with polyclonal antibodies against GFR{alpha}4 (goat AF1677; R&D Systems, Inc., Minneapolis, MN) or Ret (goat AF 482; R&D Systems, Inc.) and calcitonin (rabbit; Dako, Carpinteria, CA). Donkey secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Confocal images were taken with a Zeiss LSM5 imaging system equipped with suitable lasers and software (Zeiss, Oberkochen, Germany). Specificity of the Ret antibody was tested on thyroid sections from E18.5 Ret-KO mice (12). Sections from other WT tissues were screened for GFR{alpha}4 immunoreactivity using sections from corresponding GFR{alpha}4-KO tissues as negative and sections from WT thyroids as positive controls.

Calcitonin measurement
Thyroid tissue samples from newborn (P0-P1), approximately 3-wk- and approximately 10-month-old GFR{alpha}4-KO and WT mice were homogenized in 100 µl/10 mg tissue weight of 0.1 M HCl. The supernatant from tissue samples were frozen in liquid nitrogen until analysis. Calcitonin levels were measured by immunoradiometric assay (rat calcitonin IRMA kit; Immutopics, San Clemente, CA). Values were expressed as pictogram equivalents of rat calcitonin per milliliter. The stated detection limit of the assay was 1.0 pg/ml. No difference was observed between female and male mice, so results were pooled together.

Bone formation
For in vivo double calcein fluorescent labeling (13, 14), mice were given two injections of calcein (25 mg/kg; Sigma, St. Louis, MO) 1 wk apart: first injection at P11 and second at P18. Animals were killed 2 d after the last injection at P21, and perfused transcardially with 4% paraformaldehyde. Tibias were dissected, postfixed overnight, and embedded in methyl metacrylate. For each animal, at least five unstained nonconsecutive horizontal sections (10 µm) of undecalcified proximal tibia were analyzed using fluorescence microscopy. Mineral apposition rate was determined (from digital microscopic images) by the average distance between the two bands of calcein label using Image Pro Plus software (Media Cybernetics) by an experimenter who was blind with respect to the genotype of the samples.

Statistics
All results are expressed as mean ± SEM. Statistical significance between the genotypes was determined using a two-tailed Student t test, assuming unequal variance. Values of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of GFR{alpha}4-KO mice
To produce GFR{alpha}4-KO mice, we deleted a large part of the Gfra4 gene, using homologous recombination in embryonic stem cells (Fig. 1AGo). Chimeric mice derived from these cells were bred to C57BL/6 and 129/Sv females to establish heterozygotes. GFR{alpha}4-KO mice were born at the expected Mendelian frequency (data not shown). Lack of Gfra4 expression in the KO mice was confirmed by RT-PCR from thyroid (Fig. 1CGo). No differences were observed between WT and GFR{alpha}4-KO littermate animals in growth, gross behavior, viability, or fertility. Basic histological analysis of central nervous system, pituitary gland, and adrenal gland did not reveal differences between WT and GFR{alpha}4-deficient mice (data not shown).

Selective expression of functional Gfr{alpha}4 mRNA in juvenile mouse thyroid C cells
We showed previously by RT-PCR that mRNA encoding for the functional, GPI-anchored GFR{alpha}4 is expressed in 3-wk-old mouse thyro-parathyroid gland preparations, and by in situ hybridization that Gfra4 transcripts are present in the thyroid C cells, as well as in the parathyroid gland (9). To study which cells in the thyro-parathyroid glands express Gfra4 mRNA with the functional signal sequence encoded by exon-1a (9), we analyzed 3-wk-old WT mouse thyro-parathyroids by in situ hybridization using probes recognizing different parts of mouse Gfra4 gene. As previously reported (9), a probe recognizing the 3' part of Gfra4 (lacking exon-1a) hybridized with cells in the thyroid medulla (C cells), as well as in the parathyroid gland (Fig. 2Go, A and B) and brain (Fig. 2GGo). In contrast, hybridization with a probe specific for mRNA from exon-1a, gave a clear signal in the thyroid medulla, whereas no signal was detected in the parathyroid gland (Fig. 2Go, C and D) or in brain (Fig. 2Go, E and F, and data not shown). Lack of full-length Gfra4 mRNA in brain was also evident using RT-PCR analysis (Fig. 2HGo). These results show that, in juvenile mice, functional Gfra4-transcripts are produced in the thyroid C cells but not in the parathyroid gland or brain.


Figure 2
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  		FIG. 2. Functional Gfra4 mRNA is expressed in juvenile thyroid C cells but not in the parathyroid gland or in the brain. Shown are 3-wk-old WT mouse thyroid (A–D) and adult brain (E–G) sections hybridized with Gfra4 3' (A, B, G) and exon-1a (C–F) probes. Bright-field images A, C, and E correspond to dark-field images B, D, and F, respectively. The arrowhead points at the parathyroid gland. Scale bar in A–D, 100 µm; in E–G, 500 µm. H, RT-PCR analysis of full-length Gfra4 transcripts in different areas of adult mouse brain. One-week-old mouse thyroid was used as a positive control. The 780- and 879-bp PCR products correspond to the GPI-anchored and transmembrane forms of GFR{alpha}4, respectively. Amplification of control transcript indicated equal loading of cDNA (data not shown).

 
GFR{alpha}4 and Ret proteins are colocalized exclusively in thyroid C cells of young mice
A polyclonal antibody against mouse GFR{alpha}4 was used to determine the expression of GFR{alpha}4 protein in different tissues known to express Gfra4 mRNA (9). Clear GFR{alpha}4 immunoreactivity was seen in most (but not all) WT mouse thyroid C cells in all time points (newborn, 3-wk, adult) examined (Fig. 3Go, A–I), whereas no staining was observed in GFR{alpha}4-KO thyroid (Fig. 3JGo). In contrast, GFR{alpha}4 immunoreactivity was undetectable in all other newborn, 3-wk-old, and adult WT mouse tissues examined including the adrenal gland, testis, pituitary, parathyroid gland, and brain (data not shown). On the other hand, Ret immunoreactivity in thyroid C cells was down-regulated during postnatal development being prominent at birth (E18.5), decreased in the 3-wk-old, and virtually absent in the adult (Fig. 3Go, M–R, and data not shown). Specificity of the Ret immunostaining was demonstrated in thyroid sections from Ret-KO mice (Fig. 3Go, S–U). This postnatal decrease of Ret expression in C cells is consistent with our previous mRNA in situ hybridization results (9).


Figure 3
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  		FIG. 3. GFR{alpha}4 protein is expressed in thyroid C cells of both young and adult mice, whereas Ret is expressed in the C cells only of newborn mice. A–I, Immunostaining of newborn (A–C), 3-wk-old (D–F), and adult (G–I) WT mouse thyroid sections for GFR{alpha}4 (left, red) and calcitonin (middle, green). Yellow staining in merged images (right) represents colocalization. J–L, Thyroid C cells in adult GFR{alpha}4-KO mice identified with calcitonin staining (K) show no GFR{alpha}4 labeling (J) above background. M–R, Immunostaining of E18.5 (M–O) and adult (P–R) WT mouse thyroids for Ret (red) and calcitonin (green). S–U, Thyroid C cells from E18.5 Ret-KO mice labeled with Ret antibody show no labeling above background. Scale bar, 50 µm.

 
Expression of other GDNF family receptors in mouse thyroid gland
To analyze whether lack of GFR{alpha}4 might cause compensatory up-regulation of other GDNF family receptor mRNAs in thyroid, sections from 1-wk-old WT and GFR{alpha}4-KO mouse thyroids were hybridized with specific mouse RNA in situ probes for Gfra1, Gfra2, Gfra3, or Ret (Fig. 4Go). No expression of Gfra1 or Gfra3 mRNAs was detectable in thyroid gland of WT or GFR{alpha}4-KO mice. Ret mRNA was clearly expressed in distinct clusters of medullary thyroid cells, as reported previously (9, 15, 16), and the expression was indistinguishable between the genotypes. In contrast to the concentration of Ret (and Gfra4) expression in the medullary part of the gland, Gfra2 mRNA was found in scattered cells across the thyroid gland with similar pattern in both genotypes. Double-staining with calcitonin and GFR{alpha}2-specific antibodies indicated that GFR{alpha}2 is not expressed in the C cells (data not shown). These results show that juvenile mouse thyroid C cells express Ret and GFR{alpha}4 but not other GDNF family receptors, and that the other receptors are not up-regulated in GFR{alpha}4-KO mice to compensate for the lack of GFR{alpha}4 expression.


Figure 4
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  		FIG. 4. Lack of GFR{alpha}4 does not cause compensatory up-regulation of other GFR{alpha} or Ret mRNAs. In situ hybridization of adjacent sections of 1-wk-old WT and GFR{alpha}4-KO mouse (129/B6 background) thyroid for Ret (A–C), Gfra1 (D–F), Gfra2 (G–I), and Gfra3 (J–L). Ret mRNA is expressed in C cells with a similar intensity and pattern in WT (A) and GFR{alpha}4-KO mice (B). Gfra2 is expressed in both WT (G) and GFR{alpha}4-KO (H) thyroids in cells that are not C cells (see Results). Gfra1 (D and E) and Gfra3 (J and K) are undetectable in thyroid glands of both genotypes. Bright-field images C, F, I, and L correspond to dark-field images A, D, G, and J, respectively. Scale bar, 200 µm.

 
Normal development of thyroid C cells in GFR{alpha}4-KO mice
To study whether GFR{alpha}4 is necessary in C cell development, we immunostained thyroid sections from newborn and adult GFR{alpha}4-KO and WT littermate mice for calcitonin. The morphology and distribution of C cells were indistinguishable between the genotypes in both ages (Fig. 5Go and data not shown). Counting of the calcitonin-immunoreactive profiles from serial sections through the thyroid gland revealed no differences between GFR{alpha}4-KO and WT littermate mice at birth (WT = 580 ± 40, KO = 570 ± 150, P = 0.95, n = 3 mice of both genotypes) or in adults (WT = 2180 ± 320, KO = 2190 ± 340, P = 0.98, n = 7 mice of both genotypes, four 10-wk-old and three 16-month-old littermates).


Figure 5
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  		FIG. 5. Thyroid C cells develop normally in the absence of GFR{alpha}4. A–D, Calcitonin immunostaining of thyroid sections from adult WT (A and C) and GFR{alpha}4-KO (B and D) littermate mice in hybrid 129/B6 background. The size, morphology, and distribution of C cells, as well as the intensity of calcitonin labeling appear similar between the genotypes. Scale bars: A, 200 µm; C, 20 µm.

 
Ret and Gfra1 are coexpressed in the ultimobranchial body
Because GFR{alpha}4 was not needed for C cell development, we studied whether another GFR{alpha} would be expressed in the C cells during embryonic development. C cells originate from the ultimobranchial body, a neural crest-derived structure from which the C cell precursors migrate and invade the thyroid rudiment (17). Although Gfra1 is not expressed in thyroid C cells at birth (9) or postnatally (Fig. 4Go), in situ hybridization of E12 mouse embryos showed that the expression patterns of Ret and Gfra1 mRNAs overlap in the ultimobranchial body (Fig. 6Go). This suggests that GFR{alpha}1 and Ret are coexpressed in developing C cells during early embryonic development.


Figure 6
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  		FIG. 6. Gfra1 is coexpressed with Ret in the ultimobranchial body at E12. In situ hybridization of adjacent sagittal sections for Ret (A and B) and Gfra1 (C and D). Arrows point to the ultimobranchial body. Bright-field images B and D correspond to the dark-field images A and C. Scale bar, 100 µm.

 
Reduced thyroid calcitonin levels in newborn and juvenile but not adult GFR{alpha}4-KO mice
To study whether the lack of GFR{alpha}4 has an effect on calcitonin production in the thyroid gland, we measured thyroid calcitonin levels using an immunoradiometric assay (Fig. 7Go). Calcitonin levels in thyroid tissue were reduced by 60% in the newborn (WT = 17 ± 1 ng/ml, n = 20, KO = 7 ± 1 ng/ml, n = 18; P < 10–5) and by 45% in the juvenile GFR{alpha}4-KO mice (WT = 53 ± 4 ng/ml, n = 17; KO = 29 ± 2 ng/ml, n = 21; P < 10–5). In contrast, thyroid calcitonin levels in adult mice were similar between the genotypes (WT = 55 ± 15 ng/ml, n = 10; KO = 56 ± 15 ng/ml, n = 7; P = 0.7). This phenotype seemed to depend on genetic background because when using newborn mice in hybrid 129/B6 background, the reduction in thyroid calcitonin levels was prominent (80%, WT = 20 ± 2 ng/ml, KO = 4 ± 1 ng/ml, n = 10 in both genotypes; P < 10–5), whereas in newborn mice in B6 background, thyroid calcitonin levels were almost similar between the genotypes (WT = 13 ± 2 ng/ml, n = 10, KO = 11 ± 2 ng/ml, n = 8, P = 0.3). Serum calcitonin levels were reduced by approximately 40% in juvenile (3- to 4-wk-old) GFR{alpha}4-KO mice in hybrid 129/B6 background (WT = 71 ± 13 pg/ml, n = 11; KO = 42 ± 8 pg/ml, n = 13), although this did not reach significance (P = 0.07).


Figure 7
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  		FIG. 7. Thyroid calcitonin levels are reduced by 60% in newborn and by 45% in juvenile (~3 wk old) but are normal in adult (~10 months old) GFR{alpha}4-KO animals compared with WT controls. The data include all the mice (in 129/B6 or B6 background) analyzed. ***, P < 10–5 between the genotypes. Individual values are indicated as dots and the number of mice in each group is shown in parentheses.

 
Increased bone formation in juvenile GFR{alpha}4-KO mice
Calcitonin as well as calcitonin receptor-deficient mice display a greater mineral apposition and bone formation rate compared with their WT littermates (18). To study whether a similar defect could be seen in the GFR{alpha}4-KO mice, we assessed the bone formation by measuring the mineral apposition rate after double injection of calcein in a separate set of juvenile mice (in hybrid 129/B6 background) (Fig. 8Go). The distance between the two calcein-labeled areas (mineral apposition rate) was significantly greater in GFR{alpha}4-KO compared with WT mice (WT = 1.4 ± 0.04 µm/d, KO = 1.8 ± 0.1 µm/d, n = 9 in both genotypes, P < 0.05).


Figure 8
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  		FIG. 8. Mineral apposition is increased in GFR{alpha}4-KO mice. Double calcein labeling was performed to assess the mineral apposition rate in GFR{alpha}4-KO mice in hybrid 129/B6 background. A and B, A representative pattern of double calcein labeling in juvenile WT and GFR{alpha}4-KO mouse proximal tibia. Two calcein labels are indicated with arrows. C, Quantification of mineral apposition rate determined by the distance between the midpoint of two bands of calcein label (arrows) in WT and GFR{alpha}4-KO tibia (*, P = 0.03; n = 9 in both genotypes). Scale bar, 25 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we present evidence that GFR{alpha}4 is required for thyroid C cell function in vivo. Although development of the C cells was normal, thyroid calcitonin production was impaired in neonatal and juvenile GFR{alpha}4-KO mice.

The present study is the first to demonstrate endogenous GFR{alpha}4 expression at the protein level. Although Gfra4 mRNA is clearly detectable in several tissues and cell types in mice by in situ hybridization (9), we could detect GFR{alpha}4 immunoreactivity only in the C cells. Although correctly spliced, full-length Gfra4 mRNA is detectable by RT-PCR in pituitary intermediate lobe and neonatal adrenal medulla (9); GFR{alpha}4 protein appears to be expressed in these tissues at levels below detection by immunohistochemistry. We show in this study that, although Gfra4 mRNA is present in both thyroid C cells and in the parathyroid gland of juvenile mice, only the C cells express Gfra4 transcripts with the signal sequence encoded by exon-1a, presumably due to tissue-specific regulatory elements in Gfra4 promoter. Another mechanism that limits the production of full-length protein is a tissue-specific splicing of the short intron between exons 2 and 3 in Gfra4 (9). Inclusion of this intron produces Gfra4 transcripts with premature stop codons that may represent a significant fraction of Gfra4 transcripts in the thyroid and other endocrine organs, such as pituitary (9). Translation of these transcripts containing a functional signal sequence would produce a truncated, soluble protein that is secreted or undergo nonsense-mediated decay (19). Taken together, like in humans (3), GFR{alpha}4 expression in mice is restricted to the thyroid C cells.

A subpopulation of thyroid C cells requires Ret-signaling during embryonic development, because Ret-deficient mice have approximately 37% less calcitonin-immunoreactive cells than WT littermates at birth (9). We show that postnatal mouse thyroid C cells express mRNA for Ret and Gfra4 but not for the other Gfras (Ref.9 and present study). In addition, GFR{alpha}4 and Ret protein are colocalized in newborn and juvenile thyroid C cells. Yet, lack of GFR{alpha}4 did not affect the number of C cells in newborn or in adult mice. Furthermore, the distribution and morphology of the C cells was indistinguishable between the genotypes. Thus, GFR{alpha}4 is not necessary for the development of thyroid C cells. In addition to Gfra4 and Ret (9), we show in this study that also Gfra1 mRNA is expressed by the C cell precursors in the ultimobranchial body of E12 mouse embryos. Thus, the Ret-dependent subpopulation of C cells may require signaling through GFR{alpha}1 during embryonic development. Confirmation of this hypothesis awaits studies using GFR{alpha}1/GFR{alpha}4 double KO mice.

In contrast to the normal development of C cells, we found that newborn and 3-wk-old GFR{alpha}4-KO mice have significantly reduced thyroid calcitonin levels compared with WT mice in hybrid 129/B6 background. The variable penetrance of the calcitonin phenotype in GFR{alpha}4-KO mice, being clear in the hybrid but not in congenic B6 background, is presumably due to background effects. Similarly, background effects were suggested for the incomplete penetrance of phenotype (ptosis due to a deficit in superior cervical ganglion development) in ARTN- and GFR{alpha}3-KO mice (20). Thus, our results indicate that GFR{alpha}4 is required for the production of calcitonin during postnatal development in mice. Because no significant differences were observed between the genotypes in thyroid calcitonin levels in adult mice, the functional C cell phenotype observed in newborn and juvenile GFR{alpha}4-KO mice is unlikely to be secondary due to a developmental effect. Instead, it indicates a direct, active role for GFR{alpha}4 in modulating calcitonin production. Because GFRA4 and RET mRNAs are selectively coexpressed also in the human thyroid C cells (3), GFR{alpha}4/Ret signaling may enhance calcitonin production in humans.

Calcitonin, a 32-amino acid peptide hormone originally characterized by its potent plasma calcium-lowering effects (21), is used therapeutically to treat hypercalcemia and metabolic bone diseases, such as osteoporosis and Paget’s disease, characterized by increased bone resorption reflecting high osteoclast activity (22, 23). Yet, the physiological role of calcitonin has remained unclear until recently. Although exogenous calcitonin is known to inhibit bone resorption (24) and lower serum calcium concentration through specific receptors on the osteoclasts (25, 26), mice lacking calcitonin (13) or its receptor (18) have normal bone resorption but instead show increased bone mass due to an approximately 30% increase in bone formation. Consistent with the reduced (but not absent) calcitonin levels, we observed a similar but milder increase in bone formation in the juvenile GFR{alpha}4-KO mice.

A plausible reason for GFR{alpha}4 being required for calcitonin production only in young but not in the adult mice is suggested by the apparently higher level of Ret mRNA (9) and protein (this study) expression in newborn and juvenile C cells than in adult C cells. In contrast, GFR{alpha}4 protein expression in C cells appeared similar in developing and adult C cells. Moreover, semiquantitative RT-PCR analysis using primers that amplify the full-length Gfra4 mRNAs suggests that the relative expression of the functional, GPI-anchored isoform (27) and the dominant-negative, transmembrane isoform (28) of mouse GFR{alpha}4 is similar among newborn, juvenile, and adult thyroid C cells (data not shown).

The mechanism of how GFR{alpha}4, presumably via Ret signaling, helps to regulate thyroid calcitonin levels remains to be studied. The lower thyroid calcitonin levels in juvenile GFR{alpha}4-KO mice cannot be attributed to increased calcitonin secretion because serum calcitonin levels were reduced also (and not increased). Our preliminary data (see supplemental Fig. 1 published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) suggest that GFR{alpha}4 signaling may regulate calcitonin mRNA levels. Signal transduction pathways and mechanisms that regulate calcitonin production are poorly known. Calcitonin secretion is known to be stimulated by elevated levels of extracellular calcium [Ca2+]e via the G protein-coupled calcium-sensing receptor (CaR) on the C cells (29, 30). Calcitonin gene expression is also stimulated by elevated [Ca2+]e and CaR, presumably via [Ca2+]i-dependent activation of transcription factor TTF-1 (31). CaR activation is reported to stimulate protein kinase C via phosphatidylinositol 3-kinase signaling pathways (32), and CaR-mediated release of intracellular calcium stores is negatively regulated by protein kinase C (29). One possible mechanism how GFR{alpha}4 might regulate calcitonin synthesis could be modulation of the CaR signal transduction by Ret. For example, C cells express phosphatidylinositol 3-kinase isoforms (32) that could be synergistically activated through tyrosine kinase and G protein-coupled receptors, respectively (33). It might also be worth examining whether calcitonin production per C cell is increased in the MEN2 cancer syndrome, in which Ret signaling is abnormal due to gain-of-function mutations in RET. Our preliminary results (supplemental Fig. 2) indicate that PSPN can activate endogenous RET phosphorylation and downstream signaling in human TT cells (an established C cell line with a MEN2A mutation in RET). Because calcitonin regulation in these cells may be abnormal, future experiments on the mechanism of how PSPN regulates calcitonin levels should also use organotypic thyroid cultures or purified C cells.

GFR{alpha}4 was originally identified in chicken (34) and was shown to bind human PSPN (35). However, the endogenous ligand for chicken GFR{alpha}4 remains unclear because chicken genome lacks the ortholog of PSPN (Airaksinen, M. S., unpublished data). The chicken and the mammalian GFR{alpha}4 differ in their domain structure (9) and tissue expression patterns (36, 37), suggesting that their biological functions are also different. Thus, compared with GFR{alpha}1 and GFR{alpha}2 (1), biological function of GFR{alpha}4 may be less conserved in the vertebrate evolution.

Our results suggest that PSPN, the only known ligand for mammalian GFR{alpha}4 (3), would be required for calcitonin production, which remains to be studied using PSPN-deficient mice (8). However, Pspn mRNA levels are barely detectable in mouse thyroid in contrast to several other tissues such as adrenal gland and fat that clearly express Pspn (supplemental Fig. 3). Differently than other GDNF family ligands, PSPN does not bind to tissue matrix and, therefore, could circulate through body fluids (Bespalov, M., S. Tumova, A. Hienola, H. Rauvala, and M. Saarma, unpublished data). However, lack of good antibodies has precluded us from determining which cells produce PSPN in young mice and whether PSPN levels are adequate to support this hypothesis.

Tomac et al. (8) have reported that the PSPN-deficient mice are hypersensitive to cerebral ischemia and that neuronal cell death can be markedly attenuated by administration of recombinant human PSPN. These findings suggested that functional GFR{alpha}4 is expressed in the brain as a receptor of PSPN. However, our present results strongly suggest that functional GFR{alpha}4 is not detectable in the mouse brain. Future studies are warranted to test whether or not GFR{alpha}4-KO mice show a similar ischemia phenotype as the PSPN-KO mice.

In conclusion, we show that 1) GFR{alpha}4 protein expression is limited to thyroid C cells in mice, 2) thyroid calcitonin production is impaired in juvenile GFR{alpha}4-KO mice 3) accompanied with increased bone formation, and 4) that the expression of this phenotype correlates with the coexpression of functional GFR{alpha}4 and Ret in C cells in young mice. The present study indicates a role for endogenous GFR{alpha}-signaling to regulate calcitonin synthesis in vivo. Similar to GFR{alpha}4 in C cells, other GFR{alpha} receptors may also regulate transmitter production in other cells.


   Acknowledgments
 
We thank Kaija Berg, Eila Kujamäki, and Satu Åkerberg for technical assistance, and Raija Ikonen and Heikki Rauvala at the Viikki Transgene Unit for the morula aggregation.


   Footnotes
 
This work was supported by grants from the Academy of Finland, European Union (QLRT-2001-01000), and Sigrid Jusélius Foundation (to M.S.A. and M.S.).

Disclosure statement: P.L. has nothing to declare. M.L., J.R., M.S., and M.S.A. are coinventors on a patent (EP00977623.8).

First Published Online February 23, 2006

Abbreviations: CaR, Calcium-sensing receptor; GDNF, glial cell line-derived neurotrophic factor; GFR{alpha}, GDNF family receptor {alpha}; GPI, glycosyl-phosphatidylinositol; KO, knockout; PSPN, persephin; WT, wild type.

Received December 20, 2005.

Accepted for publication February 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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