The Role of Hoxa3 Gene in Parathyroid Gland Organogenesis of the Mouse
Department of Anatomy, Kitasato University School of Medicine (YK,YA,TN), Sagamihara, Kanagawa, and Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University (OC), Kyoto, Japan
Correspondence to: Yoko Kameda, Dept. of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555, Japan. E-mail: kameda{at}med.kitasato-u.ac.jp
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Summary |
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Key Words: Hoxa3 parathyroid thymus third pharyngeal pouch SP-1/chromogranin A connexin43lacZ transgenic mice neural crest cells apoptosis
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Introduction |
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Chromogranin A, the major secretory protein of adrenal chromaffin cells, is a member of the granin/secretogranin family of acidic glycoproteins that participate in the storage and secretion of peptide hormones and are expressed in many endocrine and neuroendocrine cells (O'Connor et al. 1983; FischerColbrie et al. 1987
; Winkler and FischerColbrie 1992
). Secretory protein-1 (SP-1) coexists with parathormone (PTH) in secretory granules of the parathyroid chief cells (Cohn et al. 1984
). SP-1 and chromogranin A are chemically similar if not identical proteins (Cohn et al. 1982
). The present study shows that SP-1 immunoreactivity appears in the parathyroid rudiment of the third pharyngeal pouch from the earliest stage of its organogenesis.
Hoxa3 belongs to the Hox family of transcription factors that play multiple roles in the segmental processes of anteroposterior patterning (Krumlauf 1994; Trainor and Krumlauf 2000
). In the Hoxa3 homozygous null mutant mouse produced by gene targeting, the thymus and parathyroid glands derived from the third pharyngeal pouch are lacking (Chisaka and Capecchi 1991
). The Hoxa3 homozygotes also show a deficiency of the common carotid artery originating from the third arch artery (Kameda et al. 2003
). However, the initial formation of the third arch artery is not disturbed; the artery degenerates at embryonic day (E) 11.5 (Kameda et al. 2002
). Therefore, the Hoxa3 gene is crucial for the development and differentiation of the third pharyngeal arch and pouch. It is unknown whether or not the initial formation of the parathyroid rudiment is affected by a lack of the Hoxa3 gene.
It has been demonstrated by studies of chickquail chimeras that the mesenchymal components of thymus lobes and parathyroid glands of avian species are derived from the ectomesenchymal neural crest cells (Le Lièvre and Le Douarin 1975). Ablation of the premigratory cardiac neural crest in chick embryos results in disrupted development of the caudal pharyngeal arches, including thymus and parathyroid hypoplasia or agenesis (Kirby and Waldo 1995
). Fate-mapping techniques using neural crest-specific transgenes have recently become available for mouse embryos (Lo et al. 1997
; Waldo et al. 1999
; Jiang et al. 2000
). In connexin (Cx) 43lacZ transgenic mice in which the promoter sequence for Cx43 linked to a lacZ reporter, the derivatives of neural crest cells are specified by IHC demonstration of ß-galactosidase expression.
The present study first clarified the organogenesis of parathyroid glands in wild-type mouse embryos using the SP-1/chromogranin A antiserum as a specific marker for parathyroid cells. Furthermore, we visualized in Cx43lacZ transgenic mice whether or not mesenchymal neural crest cells enter the parathyroid and thymus primordia to control the growth of these organs. The main purpose of this study was to examine the etiology of the parathyroid deficiency in the Hoxa3-null mutants. Migrations of neural crest cells into the pharyngeal arches and apoptosis of the third pouch endoderm were estimated in Hoxa3 homozygous mutants in comparison with wild-types.
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Materials and Methods |
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For histological study, the specimens were fixed in Bouin's solution or 8% paraformaldehyde (PFA) in phosphate buffer (PB) for 2448 hr, embedded in paraffin, and then serially sectioned in the frontal or sagittal plane at a thickness of 5 µm. Selected sections were stained with hematoxylineosin to help determine morphological orientation.
TUNEL Assay
To visualize apoptotic nuclei, sections fixed in 4% or 8% PFA in PB were stained by the terminal transferase dUTP-biotin nick-end labeling (TUNEL) technique according to the manufacturer's instructions (ApopDETEK In Situ Cell Death Assay kit; DAKO, Carpinteria, CA). Specificity controls included the alternative omission of the biotin-dUTP, of the terminal deoxynucleotidyl transferase (TdT), or of the streptavidinhorseradish peroxidase conjugate. No labeling was detected in any control sections.
Immunohistochemistry
IHC staining was carried out by the streptavidinbiotinperoxidase method or the peroxidaseantiperoxidase (PAP) method as described previously (Kameda et al. 1998). The following primary antibodies were employed: the monoclonal anti-ß-galactosidase antibody and the polyclonal anti-SP-1/chromogranin A and anti-human protein gene product (PGP) 9.5 antisera. The SP-1/chromogranin A antiserum was purchased from Incstar (Stillwater, MN) and used at a dilution of 1:500. The PGP 9.5 antiserum was purchased from UltraClone (Isle of Wight, UK) and used at a dilution of 1:600. The ß-galactosidase antibody was purchased from Promega (Madison, WI) and used at a dilution of 1:300. Control reactions included replacing the primary antibodies with normal (non-immune) rabbit or mouse Ig and omission of the primary antibodies. In addition, for ß-galactosidase immunoreactivity non-transgenic embryos were examined in parallel as controls. All control reactions were negative.
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Results |
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Subsequently, the time course of parathyroid/thymus primordia development was determined in wild-type embryos. At E 12.0, the thymus rudiment originating from the third pharyngeal pouch became clear and extended in a caudal direction. The thymus still displayed a follicle structure in which the epithelium had many mitotic figures. The parathyroid rudiment was attached to the cranial part of the thymus (Figure 4A) . At E 12.5, the thymus rudiment was significantly increased in size and formed a solid cell cluster. It began to move towards the anterior thoracic cavity. The parathyroid rudiment was localized on the top of the thymus (Figure 4B). At E 13.5, owing to a caudal migration of the thymus, the parathyroid rudiment contiguous with the top of the thymus was situated at the side of the thyroid and ultimobranchial glands (Figure 4C). At E 14.5, the parathyroid joined with the lateral side of the thyroid gland and was completely separated from the thymus, showing a normal position in mature mice (Figure 4D).
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Discussion |
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Hoxa3 mRNA is expressed in the third pharyngeal pouch endoderm (Manley and Capecchi 1995). The present study demonstrated that the Hoxa3 gene is required for initiation of parathyroid rudiment formation. In wild-type mouse embryos, the differentiation of the third pharyngeal pouch began at E 11.5. The parathyroid rudiment immunoreactive for SP-1/chromogranin A was formed in the cranial dorsal part of the third pouch endoderm and could be discriminated from the thymus at this stage of development. In Hoxa3 homozygous mutants, however, the third pharyngeal pouch still remained in high columnar epithelium and bulges for parathyroid and thymus primordia were not formed at E 11.5. No SP-1/chromogranin A-immunoreactive cells were detected in the third pharyngeal pouch. Therefore, the third pharyngeal pouch failed to differentiate and finally disappeared in the Hoxa3 homozygotes. The intrinsic capability of the third pharyngeal pouch to form the parathyroid glands was affected by the lack of the Hoxa3 gene.
Some candidates for signaling molecules involved in the parathyroid organogenesis have been reported. Glial cells missing (Gcm) 2, a mouse homologue of Drosophila Gcm, is the transcription factor demonstrated in the parathyroid gland in late stages of fetal development (Kim et al. 1998). Gcm2-deficient mice lack parathyroid glands but show normal thymus development (Günther et al. 2000
). Gcm2 expression begins at E 9.5 in the caudal pharyngeal pouches and is progressively restricted to the third pharyngeal pouch endoderm (Gordon et al. 2001
). In contrast to Gcm expression, SP-1/chromogranin A immunoreactivity was localized only in the parathyroid rudiment in the pharyngeal pouches. Pax1 and Pax9 are closely related members of the paired box gene family. Pax1 is expressed in the third pharyngeal pouch and also in thymic epithelial cells (Wallin et al. 1996
). Loss of Pax1 function results in a hypoplastic thymus due to a deficiency in thymocyte development (Wallin et al. 1996
; Su et al. 2001
). Although hypoplastic parathyroids have also been reported in Pax1 mutant embryos, it is difficult to precisely identify the degenerative organs by hematoxylineosin staining (Su et al. 2001
). Pax9 mutant mice lack the derivatives of the third and fourth pouches, i.e., thymus, parathyroid, and ultimobranchial glands (Peters et al. 1998
). Eyes absent (Eya) 1 gene is expressed in thymus, parathyroid, and ultimobranchial glands during development, and the organ primordia for these structures fail to form in Eya1/ embryos (Xu et al. 2002
). In Hoxa3 mutants, Pax1 is downregulated in the third pharyngeal pouch (Manley and Capecchi 1995
) and Gcm expression is absent (Ellis S. et al., quoted in Su et al. 2001
). In Eya1/ pouch endoderm, Hoxa3, Pax1, and Pax9 expression is not affected (Xu et al. 2002
). It has been suggested that Gcm2 expression in the parathyroid glands is regulated by the Hoxa3Pax1/9Eya1 pathway (Xu et al. 2002
). Therefore, Hoxa3 seems to be a key regulator in differentiation of the third pouch endoderm.
In human 22q11 deletion syndromes, hypoplastic or aplastic thymus and parathyroid glands are characteristic phenotypic abnormalities (Scambler 2000). Tbx1 is a T-box transcription factor that lies in the 22q11 locus, and Tbx1-null homozygotes lack the thymus and parathyroid glands (Jerome and Papaioannou 2001
). Fgf8 mutant mice display phenotypes seen in human 22q11 deletion syndromes, including thymic and parathyroid aplasia and hypoplasia (Frank et al. 2002
). The relationships of these genes to Hoxa3 during third pharyngeal pouch development remain to be elucidated.
In Hoxa3 homozygotes crossed with Cx43lacZ transgenic mice, ectomesenchymal neural crest cells immunoreactive for ß-galactosidase densely populated the pharyngeal arches as in wild-type embryos. The migration of mesenchymal neural crest cells into the third arch was not disturbed in the Hoxa3 homozygotes. In avian species, neural crest cells invade the thymus and parathyroid glands and give rise to glandular connective tissue (Le Lièvre and Le Douarin 1975). On the other hand, the present study demonstrated in mouse embryos that neither the endodermal epithelium of the third pharyngeal pouch nor the parathyroid and thymus primordia received the neural crest cells. Furthermore, it has been shown in the mouse by marking the Wnt1-Cre and R26R genes that the mesenchymal neural crest cells surround but do not invade the thymus parenchyma (Jiang et al. 2000
). The parathyroid, thymus, and ultimobranchial primordia develop from the pharyngeal pouches and migrate to their final destinations. These organs are densely enclosed by mesenchymal neural crest cells during movement to their locations. The neural crest cells distributed in the pharyngeal arches may be involved in the organ primordia migration in the mouse embryos. In addition, the condensation of mesenchymal neural crest cells during development of pharyngeal pouch-derived organs appears to be accompanied by upregulation of the expression of several growth factors and transcription factors (Clouthier et al. 2000
). Appropriate interactions between the pouch epithelium and adjacent neural crest-derived mesenchyme may be necessary for normal development of the parathyroid, thymus, and ultimobranchial glands. Hoxa3 mRNA is expressed in the neural crest cells populating the third pharyngeal arch (Watari et al. 2001
). Absence of Hoxa3 signaling appears to affect the proliferation and differentiation of postmigratory neural crest cells in the third arch.
The endodermal epithelia of the pharyngeal pouches in both wild-type and homozygous embryos showed many apoptotic cells. The differentiation and growth of the pharyngeal pouch-derived organs appear to be controlled by apoptosis. In particular, increased apoptotic cells were detected in the third pouch epithelium showing delayed development in the E 11.512.0 mutants. Therefore, the third pouch endoderm is unable to differentiate in the absence of the Hoxa3 gene and is eventually lost by apoptosis. Although a parathyroid rudiment was never detected, rudimentary thymus lobes were rarely encountered in the null mutants at E 12.514.5. The thymus is formed by the third pouch endoderm fused with the ectodermal cells of the third cleft (Cordier and Haumont 1980). The ectodermal cells may be unaffected in the Hoxa3 mutants and may have the ability to form unstable thymus lobes.
In contrast to the third pharyngeal pouch, the development and differentiation of the fourth pouch were normal in Hoxa3-null mutants. The bilateral ultimobranchial bodies derived from the fourth pharyngeal pouch were always formed and joined with the thyroid glands at E 13.5. At E 14.5, the C-cells immunoreactive for PGP 9.5 began to disperse within the thyroid parenchyma in the Hoxa3 mutants as well as wild-types (Kameda et al. unpublished data). In both wild-type and mutant mice, however, SP-1/chromogranin A immunoreactivity appeared in the C-cells at late stages of fetal development. Furthermore, the appearance of calcitonin immunoreactivity in the C-cells was very late, i.e., at birth. In accordance with our data, Xu et al. (2002) have reported that calcitonin immunoreactivity is first detected in newborn mice.
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Footnotes |
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