Available online at www.sciencedirect.com Developmental Biology 314 (2008) 236–247 www.elsevier.com/developmentalbiology FRS2α2F/2F mice lack carotid body and exhibit abnormalities of the superior cervical sympathetic ganglion and carotid sinus nerve ⁎ Yoko Kameda a, , Masataka Ito b, Toshiyuki Nishimaki a, Noriko Gotoh c a Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555, Japan b Department of Anatomy, National Defense Medical College, Tokorozawa 359-8513, Japan c Division of Genetics, Institute of Medical Science, University of Tokyo, Minato-ku 108-8639, Japan Received for publication 25 May 2007; revised 26 November 2007; accepted 4 December 2007 Available online 8 December 2007 Abstract The docking protein FRS2α is an important mediator of fibroblast growth factor (FGF)-induced signal transduction, and functions by linking FGF receptors (FGFRs) to a variety of intracellular signaling pathways. We show that the carotid body is absent in FRS2α2F/2F mice, in which the Shp2-binding sites of FRS2α are disrupted. We also show that the carotid body rudiment is not formed in the wall of the third arch artery in mutant embryos. In wild-type mice, the superior cervical ganglion of the sympathetic trunk connects to the carotid body in the carotid bifurcation region, and extends thick nerve bundles into the carotid body. In FRS2α2F/2F mice, the superior cervical ganglion was present in the lower cervical region as an elongated feature, but failed to undergo cranio-ventral migration. In addition, few neuronal processes extended from the ganglion into the carotid bifurcation region. The number of carotid sinus nerve fibers that reached the carotid bifurcation region was markedly decreased, and baroreceptor fibers belonging to the glossopharyngeal nerve were absent from the basal part of the internal carotid artery in FRS2α2F/2F mutant mice. In some of the mutant mice (5 out of 14), baroreceptors and some glomus cells were distributed in the wall of the common carotid artery, onto which the sympathetic ganglion abutted. We propose that the sympathetic ganglion provides glomus cell precursors into the third arch artery derivative in the presence of sensory fibers of the glossopharyngeal nerve. © 2007 Elsevier Inc. All rights reserved. Keywords: FRS2α2F/2F knockout mice; Carotid body; Glomus cells; Superior cervical sympathetic ganglion; Carotid sinus nerve; Baroreceptors; Glossopharyngeal nerve; Thymus Introduction phosphatase Shp2. Disruption of the FRS2α results in embryonic lethality at embryonic day (E)7.0–7.5 due to The docking protein FRS2α has been implicated as an multiple defects in FGF-mediated signaling (Hadari et al., important mediator of FGF-induced signal transduction, and 2001). Mice carrying a targeted disruption of the Shp2- functions by providing a link between FGFRs and a variety of binding sites of FRS2α (FRS2α2F/2F) are viable until E18.5. intracellular signaling pathways (Hadari et al., 2001). FRS2α FRS2α2F/2F mice exhibit severely impaired cerebral cortex plays a key role in FGFR-dependent signaling pathways that development (Yamamoto et al., 2005). Thus, the Shp2-binding are crucial for cell proliferation, migration, survival and early sites of FRS2α appear to be required for the maintenance of embryonic development (see Eswarakumar et al., 2005 for neuronal progenitor cells and neurogenesis in the cerebral reviews). FRS2α contains four binding sites for the adaptor cortex. FRS2α2F/2F embryos are also defective in eye develop- protein Grb2, and two binding sites for the protein tyrosine ment (Gotoh et al., 2004a). FGF is a key regulator of eye formation, and appear to cooperate with bone-morphogenetic proteins (BMPs) in the regulation of early eye development (Lovicu and McAvoy, 2005). ⁎ Corresponding author. Fax: +81 42 778 8441. The carotid body is a chemosensory organ that is sensitive to E-mail address: [email protected] (Y. Kameda). arterial hypoxia, hypercapnia, and acidity. It plays a role in the 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.12.002 Y. Kameda et al. / Developmental Biology 314 (2008) 236–247 237 regulation of the respiratory system. The carotid body is densely formation is arrested in the Mash1-null mice, most likely due to innervated by the carotid sinus nerve, a sensory branch of the the absence of the superior cervical ganglion that supplies glossopharyngeal nerve, and also by sympathetic fibers (the progenitors to the carotid body rudiment. ganglioglomerular nerve) from the superior cervical ganglion of In the present study, we examined carotid body formation in the sympathetic trunk (Verna, 1979). The typical murine carotid the FRS2α2F/2F mice, in which the location of the superior body makes connections with the superior cervical ganglion. cervical ganglion of the sympathetic trunk and the distribution There are two types of cells in the carotid body, glomus cells of the carotid sinus nerve deviate from wild-type embryos. (also called chief cells or type I cells) and sustentacular cells (also called type II cells). Glomus cells have a neuroendocrine Materials and methods phenotype, and express the neuronal markers, TuJ1, PGP 9.5, neuropeptide Y and enkephalins (Kobayashi et al., 1983; Animals Kameda et al., 2002). These cells also contain biogenic amines, such as serotonin and noradrenaline, and exhibit intense, Wild-type, heterozygous, and homozygous FRS2α2F/2F mice were obtained 2F/+ positive immunoreactivity for tyrosine hydroxylase, rate-limit- from intercrosses of FRS2α mice, which were maintained on a Swiss- ing enzyme involved in catecholamine synthesis. Sustentacular Webster background (Gotoh et al., 2004a). E0.5 was designated as of noon on the day a copulation plug was identified. The genotype of the mice was obtained cells are of the glial lineage and contain aggregates of by PCR analysis using the previously described primers (Gotoh et al., 2004a). intermediate filaments. They express the glial markers, glial For histological analysis, specimens were incubated in Bouin's solution or fibrillay acidic protein (GFAP), S100 protein, and vimentin 8% paraformaldehyde (PFA) in phosphate buffer (PB) for 24 h, embedded in (Kameda, 1996, 2005). Sustentacular cells extend long paraffin and then serially sectioned along the cross, frontal, or sagittal planes at a μ – processes that surround individual glomus cells or groups of thickness of 5 m. Selected sections were stained with hematoxylin eosin to determine morphological orientation. glomus cells. The carotid body rudiment forms in the wall of the third arch Immunohistochemistry artery as a condensation of mesenchymal cells (Kondo, 1975; Kameda, 1994). In mouse embryos, the neuronal process of the Immunohistochemical staining was carried out using the streptavidin– superior cervical ganglion of the sympathetic trunk envelops the biotin–peroxidase method, as previously described (Kameda, 1996). The carotid body rudiment at E13.0. Starting at E13.5, neuronal following antibodies were used, as indicated: monoclonal anti-TuJ1 (1:500 dilution) (Berkeley Antibody Company, Richmond, CA), which recognizes the progenitor cells derived from the superior cervical ganglion neuron-specific class III β-tubulin isotype; monoclonal anti-neurofilament (NF) enter the rudiment and differentiate into glomus cells (Kameda 160 (1:200 dilution)(clone NN18; Sigma, Saint Louis, Missouri), polyclonal et al., 2002; Kameda, 2005). It has been proposed that early anti-human protein gene product (PGP) 9.5 (1:200 dilution) (Dako, Carpinteria, development of the carotid body depends on reciprocal CA); polyclonal anti-serotonin (5-HT) and polyclonal anti-bovine adrenal inductive interactions between the carotid body rudiment and tyrosine hydroxylase (TH) (Chemicon, El Segundo, CA); polyclonal anti- neuropeptide Y (UCB Bioproducts, Brussels, Belgium); polyclonal anti- the surrounding superior cervical ganglion. chromogranin A (Yanaihara Inst., Shizuoka, Japan); polyclonal anti-FGFR-2 Hoxa3 belongs to the Hox gene family, which regulates the antibody, which was directed against the cytoplasmic domain of the receptor; formation of the mammalian body plan along the antero- and polyclonal anti-FRS2. Anti-5-HT, anti-TH, anti-neuropeptide Y, and anti- posterior axis (Trainor and Krumlauf, 2000). In Hoxa3-null chromogranin A antisera were used at dilutions of 1:500–1:1000. Anti-FGFR-2 mice, the thymus and parathyroid glands, which are derived and anti-FRS2 antisera (Sigma) were used at dilutions of 1:500 and 1:200, respectively. from the third pharyngeal pouch, are absent and the common The number of proliferating cells in the superior cervical ganglion was carotid artery, which is derived from the third arch artery, is quantified using a monoclonal antibody for proliferating cell nuclear antigen bilaterally missing or very short (Kameda et al., 2003). Since (PCNA), which reacts with proliferating cells in a wide range of normal tissues. the third arch artery degenerates at E11.5, the carotid body The-PCNA antibody was purchased in pre-diluted form from Dako. primordium is never formed in the Hoxa3-null mutants (Kameda et al., 2002). The superior cervical ganglion, however, TUNEL assay for apoptosis shows hyperplastic features in these mutant mice. – – To visualize apoptotic nuclei, tissue sections were subjected to staining using The basic helix loop helix (bHLH) transcription factor a terminal transferase dUTP-biotin nick-end labeling (TUNEL) kit (ApopDE- Mash1