Postotic and preotic cranial neural crest cells differently contribute to thyroid development

Author’s Accepted Manuscript Postotic and preotic cranial neural crest cells differently contribute to thyroid development Kazuhiro Maeda, Rieko Asai, Kazuaki Maruyama, Yukiko Kurihara, Toshio Nakanishi, Hiroki Kurihara, Sachiko Miyagawa-Tomita www.elsevier.com/locate/developmentalbiology

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S0012-1606(15)30256-6 http://dx.doi.org/10.1016/j.ydbio.2015.10.026 YDBIO6907

To appear in: Developmental Biology Received date: 9 January 2015 Revised date: 17 October 2015 Accepted date: 22 October 2015 Cite this article as: Kazuhiro Maeda, Rieko Asai, Kazuaki Maruyama, Yukiko Kurihara, Toshio Nakanishi, Hiroki Kurihara and Sachiko Miyagawa-Tomita, Postotic and preotic cranial neural crest cells differently contribute to thyroid d e v e l o p m e n t , Developmental Biology, http://dx.doi.org/10.1016/j.ydbio.2015.10.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Postotic and preotic cranial neural crest cells differently contribute to thyroid development

Kazuhiro Maedaa, Rieko Asaia,c, Kazuaki Maruyamac, Yukiko Kuriharac, Toshio Nakanishia, Hiroki Kuriharac, Sachiko Miyagawa-Tomitaa,b*

a

Department of Pediatric Cardiology, Tokyo Women’s Medical University, 8-1

Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan b

Division of Cardiovascular Development and Differentiation, Medical Research

Institute, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan c

Department of Physiological Chemistry and Metabolism, Graduate School of

Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

*Correspondence to Sachiko Miyagawa-Tomita, present address: Dept of Veterinary Technology, Yamazaki Gakuen University, 4-7-2 Minami-Osawa, Hachioji, Tokyo 192-0364, Japan.

E-mail: [email protected] or [email protected]

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Abstract Thyroid development and formation vary among species, but in most species the thyroid morphogenesis consists of five stages: specification, budding, descent, bilobation and folliculogenesis.

The detailed mechanisms of these stages have not

been fully clarified. During early development, the cranial neural crest (CNC) contributes to the thyroid gland. The removal of the postotic CNC (corresponding to rhombomeres 6, 7 and 8, also known as the cardiac neural crest) results in abnormalities of the cardiovascular system, thymus, parathyroid glands, and thyroid gland. To investigate the influence of the CNC on thyroid bilobation process, we divided the CNC into two regions, the postotic CNC and the preotic CNC (from the mesencephalon to rhombomere 5) regions and examined. We found that preotic CNC-ablated embryos had a unilateral thyroid lobe, and confirmed the presence of a single lobe or the absence of lobes in postotic CNC-ablated chick embryos. The thyroid anlage in each region-ablated embryos was of a normal size at the descent stage, but at a later stage, the thyroid in preotic CNC-ablated embryos was of a normal size, conflicting with a previous report in which the thyroid was reduced in size in the postotic CNC-ablated embryos. The postotic CNC cells differentiated into connective tissues of the thyroid in quail-to-chick chimeras. In contrast, the preotic CNC cells did not differentiate into connective tissues of the thyroid. We found that preotic CNC cells encompassed the thyroid anlage from the specification stage to the descent stage. Finally, we found that endothelin-1 and endothelin type A

receptor-knockout mice and bosentan (endothelin receptor antagonist)-treated chick embryos showed bilobation anomalies that included single-lobe formation. Therefore, not only the postotic CNC, but also the preotic CNC plays an important role in thyroid morphogenesis.

Keywords: thyroid development; bilobation; cranial neural crest; quail-chick chimera; endothelin signal

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Introduction The thyroid gland is the earliest endocrine gland to appear during development. Although the gland, as an organ synthesizing thyroid hormones, is evolutionarily well conserved in all vertebrates, its shape and size vary among the species (Pinter, 2000). The thyroid gland consists of two cell types, follicular cells synthesizing thyroxin hormones (T3 and T4) and parafollicular calcitonin-producing cells (C-cells) (De Felice and Di Lauro, 2004; Nilsson and Fagman, 2013). These cells originate from two distinct regions of the pharyngeal endoderm: the median thyroid anlage, which forms the thyroid follicles, originates from the foregut endodermal floor at the region between the first and second pharyngeal arches (PAs), while the lateral anlages of the caudal pharyngeal pouches generate the ultimobranchial bodies (UBs) that develop into parafollicular C-cells (Grevellec and Tucker, 2010; O'Rahilly, 1983; Polak et al., 1974; Fagman and Nilsson, 2010; Manley and Capecchi, 1995; Nilsson and Fagman, 2013; Pinter, 2000). It has been proposed that the morphogenetic process of the thyroid gland is composed of five stages: specification (placode), budding (diverticulum), descent (downward migration), bilobation (lateral expansion), and follicle formation (final localization). The median anlage (placode) is specified as a thickened epithelium in the midline-ventral pharyngeal floor close to the aortic sac as the specification stage. At the budding stage, the anlage forms the thyroid diverticulum by invaginating into the pharyngeal mesenchyme, and then it starts to move caudally toward the base of the neck. The diverticulum expands laterally to form the bilobed structure with the isthmus along the third PA arteries. Finally, the bilateral lobes are located along the route of the paired third PA arteries (common carotid arteries in adults), and they mature and form a number of follicles after fusion with the symmetrical lateral UBs at the bilobation stage (Fagman and Nilsson, 2010; Pinter, 2000; Grevellec and Tucker 2010; Neves et al., 2012; Van Vliet, 2003; Grevellec and Tucker, 2010). In birds, fish, amphibians, and reptiles, however, the UBs are not incorporated into the thyroid glands and thus exist as independent structures (Alt et al., 2006; Grevellec and Tucker, 2010; Pinter, 2000). Although the mechanisms underlying each developmental stage remain to be elucidated, the bilobation and the final location of the thyroid are thought to be ascribable to the third PA arteries since the thyroid primordium elongates horizontally along the course of the paired third PA arteries and the bilateral thyroid becomes located at a site in close contact with the arteries. Thus it has been speculated that the third PA arteries might

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provide guidance cues in thyroid progenitors and serve as guidance tracks (Fagman and Nilsson, 2010; Lania et al., 2009). Neural crest (NC) cells are multipotential stem and migratory cell populations that derive from the dorsal part of the neural tube during development. It has been well established that cranial NC (CNC) (from the caudal diencephalon to the rhombencephalon) cells play a predominant role in the development of various structures of the face, neck and cardiovascular system (Bronner and LeDouarin, 2012; Minoux and Rijli, 2010). Several avian studies have shown that CNC cells

contribute to thyroid connective tissue and C-cells of the UBs (Bockman and Kirby, 1984; Le Lièvre and Le Douarin, 1975; Polak et al., 1974). The postotic CNC, which is a subpopulation of the CNC and is the region between the mid-otic placode and the caudal border of somite 3, corresponding to rhombomeres (r) 6, 7 and 8 (r6–r8), is well known as the cardiac neural crest.

It contributes to morphogenesis of the

cardiovascular system, thymus, parathyroid glands, and thyroid gland (Bockman and Kirby, 1984; Bockman et al., 1987; Kirby et al., 1983; Nishibatake et al., 1987). Postotic CNC cells form the aorticopulmonary septum separating the outflow tract into the aorta and pulmonary trunk, and differentiate into smooth muscle cells of the third, fourth and sixth PA arteries. Postotic CNC-ablated studies have resulted in failure of the aorticopulmonary septum to form, a charactaristic known as persistent truncus arteriosus (PTA), and posterior PA artery anomalies (Le Lièvre and Le Douarin, 1975; Bockman et al., 1987; Kirby et al., 1983; Nishibatake et al., 1987). In addition, ablation of the postotic CNC over somites 1 to 5 has revealed that the thymus, the parathyroids, and the thyroid glands are hypoplastic or aplastic (Bockman and Kirby, 1984). Therefore, the postotic CNC is considered to play an important role in morphogenesis of not only the cardiovascular system, but also the endocrine glands. Meanwhile, the preotic CNC, which is a part of the CNC and derives from the mesencephalon to r5 (mes–r5), contributes to the craniofacial morphogenesis (Dupin et al., 2010; Kontges and Lumsden, 1996; Minoux and Rijli, 2010). Recently, we observed that the preotic CNC cells also migrate into the heart and differentiate into smooth muscle cells of the coronary artery, which are involved in endothelin (Edn) signaling (Arima et al., 2012). In addition, we found that the preotic CNC cells were scattered in the right ventricle and represented a non-cardiomyocyte population. To investigated the influence of each CNC region on thyroid bilobation process, therefore, we divided the CNC into two regions, the postotic CNC and the preotic CNC (from the mesencephalon to rhombomere 5) regions and examined.

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In this study, we first made a timetable of chick thyroid morphogenesis and confirmed the relationship between the thyroid anlage and vessels during chick embryonic development. Then we compared the distribution patterns of postotic (r6–r8) and preotic (mes–r5) CNC cells using quail-chick chimeras during thyroid development and examined the thyroid defect in each of the CNC-ablated embryos. The postotic CNC cells were distributed far from the thyroid anlage at the late descent stage HH24, but they contributed to formation of the thyroid organ and UBs at the follicle formation stage HH35. On the other hand, preotic CNC cells surrounded the thyroid anlage from the late specification stage HH17 to the descent stage HH21, but they did not differentiate into thyroid tissues. In ablation studies, postotic CNC-ablated embryos exhibited a bilateral or unilateral defect of the thyroid lobes, whereas preotic CNC-ablated embryos developed a unilateral thyroid lobe. Our findings thus suggest that postotic and preotic CNC cells have distinct roles in thyroid development; bilobation of thyroid formation might not be associated with the third PA arches; and preotic CNC cells might influence thyroid morphogenesis, especially bilobation.

Finally, we examined the thyroid glands in

endothelin-1 (Edn1) and endothelin type A receptor (Ednra) knockout mice and chick embryos treated with bosentan (endothelin receptor antagonist). We found that these knockout mice and treated chick embryos had a unilateral thyroid lobe. Endothelin signaling might have an influence on the postotic and preotic CNC for thyroid development. Therefore, we herein propose a new role for the preotic CNC in thyroid bilobation during development.

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Materials and Methods Animals All animal experiments were approved by the Tokyo Women’s Medical University and University of Tokyo Animal Care and Use Committee, and were performed in accordance with the institutional guidelines. Fertilized Hojuran chicken (Gallus

gallus) eggs and Japanese quail (Coturnix coturnix japonica) eggs were obtained from Shiroyama Keien Farms (Tochigi, Japan) and the Motoki Hatchery (Saitama, Japan), respectively, and were then incubated in a humidified atmosphere at 37°C until the embryos reached appropriate stages.

Edn1 knockout (Edn1–/–) mice (Kurihara et al., 1994) and Ednra knockout (EdnraEGFP/EGFP, EGFP knock-in) mice (Asai et al., 2010) have been described previously. Mutant mice were maintained on a mixed C57BL/6J_ICR background and were housed in an environmentally controlled room at 23±2°C, with a relative humidity of 50–60% and under a 12-h light: 12-h dark cycle. The embryonic ages were determined by timed mating with the day of the plug detection being embryonic day (E) 0.5. Edn1 and Ednra knockout mouse fetuses were obtained at E18.5. Immunostaining and histology Immunostaining for whole-mounts and sections was basically performed as described previously (Metzger et al., 2008). For whole-mount immunostaining, quail-to-chick chimeras were harvested at the appropriate stages and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 1-2 hours at 4°C. After washing in PBS, the specimens were dehydrated in graded methanol and then stored in 100% methanol overnight at -20°C. They were then incubated in 5% H2O2 in methanol for 5 hours at room temperature, rehydrated in graded methanol, and rinsed twice in 100% PBT (1% TritonX-100 in PBS). After blocking twice in PBTM (2% dry non-fat milk in PBT) for 1 hour at room temperature, the samples were incubated with anti-quail nuclear antibody (QCPN) (Developmental Studies Hybridoma Bank; 1:100 in PBTM for 2 days at 4°C). Any excess primary antibody was rinsed with PBTM and then the samples were incubated with HRP-conjugated anti-mouse IgG antibody (Vector Laboratories; 1:200) overnight at 4°C. The signals were visualized with 3,3’-diaminobenzidine tetrahydrochloride hydrate (DAB; Sigma-Aldrich, Japan) as a chromogen substrate. The stained embryos were transferred in a mixture of glycerol in PBS.

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Paraffin sections (10 μm) were treated by boiling in 0.01 M sodium citrate (pH 6.0) before incubating with the primary antibody. The sections were then incubated with anti-QCPN (1:30), anti-Nkx2.1 (also known as thyroid transcription factor 1 /Titf1) (Biopat; 1:8000), anti-calcitonin antibodies (Abcam; 1:150), anti-proliferating cell nuclear antigen (PC10) (Abcam; 1:300), and TUNEL staining for apoptosis according to the protocol of the manufacturer (Promega) (Poelmann and Gittenberger-de

Groot,

1999).

Biotinylated

secondary

antibodies

(Vector

Laboratories; 1:200) were applied to the sections for 1 hour at room temperature. Immunoreactivity was detected using a VECTASTAIN ABC Elite Standard Kit (Vector Laboratories) and DAB, or streptavidin-Alexa Fluor 488 or 546 (Molecular Probes; 1:200). Fluorescence images were obtained using a computer-assisted confocal microscope. Standard staining was performed with hematoxylin-eosin (HE), and periodic acid-Schiff (PAS) staining was used for colloid-filled follicles. Three-dimensional (3D) reconstructions Serial sections stained with HE or QCPN antibody were examined for reconstruction (Arima et al., 2012). Digital images of the stained sections were loaded into the Amira software program (Visage Imaging Inc). We investigated the thyroid anlage form and volume. The average volume of the control thyroid anlage at the bilobation stage was calculated on both sides of the lobes. Quail-to-chick chimera and CNC-ablated embryos Quail-to-chick chimeras and the ablation of the premigratory CNC were carried out as described previously (Arima et al., 2012; Miyagawa-Tomita et al., 1991; Waldo et al., 1998). Chick and quail embryos were used at 6-8 somite stages for premigratory postotic CNC (r6–r8), and at 5-7 somite stages for premigratory preotic CNC (mes–r5). The stages of the host chicken and donor quail embryos were matched as closely as possible. The eggs were windowed and embryos were visualized either with 0.02% Neutral red film in 1% agar or with India ink (black ink) (Rotring, Germany) diluted 1:20 in saline solution and injected into the subgerminal cavity. The vitelline membrane was torn. Ablation of the bulk of the neural tube of chick embryos was performed using an electrolytically sharpened tungsten needle or electrical cautery. When the postotic CNC ablation was performed completely, 100% of the embryos showed outflow tract defects of the heart, which is characteristic of PTA. Therefore, we used the samples showing PTA in the heart for the examinations. The region was grafted with the corresponding

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bilateral neural folds containing premigratory CNC cells from quail embryos. Sham-operated and control embryos were stained with Neutral red or ink-injected. Then sham-operated embryos were prepared by tearing the vitelline membrane and control embryos were not torn. The eggs of CNC-grafted, CNC-ablated, sham-operated, and control embryos were sealed with cellophane tape and reincubated. For the partial rescue of preotic CNC ablation, the bulk of the chick neural tube of chick embryos from the midbrain to somites 2 or 3 were excised and quail neural folds from somites 1 to 2 or 3 was transplanted to the host chick embryos orthotopically. Ink injection Chick embryos at Hamburger and Hamilton (HH) stage 23 (Hamburger and Hamilton, 1951) were harvested and rinsed in heparinized PBS. To visualize the PA artery formation, India ink (Kiwa-guro, Sailor, Japan) was gently injected into the heart using a glass micropipette (Arima et al., 2012; Watanabe et al., 2010). The samples were fixed in 4% PFA in PBS, dehydrated, and immersed in BABB (1:2 benzyl alcohol to benzyl benzoate). In-ovo fluorescent dye injection Fluorescent dye injection was performed on chick embryos at 6-7 somite stages. CM-DiI (Molecular Probes) at 2.0 mg/ml in N,N-dimethylformamide (Wako) diluted 1: 1 in tetraglycol (Sigma) to yield working solutions of 1.0 mg/ml was used (Tirosh-Finkel et al., 2006). The dye was injected into the postotic CNC from the 1stand 2nd-somites in the control embryos. After preotic CNC ablation of chick embryos, the dye was injected into the same somites. These specimens were harvested at the appropriate stages and fixed in 4% PFA in PBS at room temperature overnight. After washing in PBS, they were immersed in 2.5% and 5% 1,4-Diazabicyclo [2.2.2]octane (Sigma- Aldrich, Japan) in PBS. Pharmacological inactivation of endothelin signaling in-ovo Thirty microliters of olive oil was dropped into the shell membrane of the chicken eggs at 48 hours of incubation (Kempf et al., 1998). In the treated group, the embryos were administered bosentan (Actelion, Ltd.) suspended in oil at 5 mg/ml. The controls were given oil only. The embryos were collected at the bilobation stage HH37 and examined histologically.

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Results

Timetable of the onset of chick thyroid morphogenesis and relationship between the thyroid anlage and vessels of chick embryos during development The process of thyroid gland morphogenesis has been described using five steps in humans, mice and zebrafish (Supplementary Table S1) (Alt et al., 2006; De Felice and Di Lauro, 2004; Fagman et al., 2006; Jiang et al., 2000). In the present study, therefore, we examined these five stages of thyroid morphogenesis in chick embryos (n=55) using immunohistochemistry and the 3D images (Guo et al., 1991; Knospe et al., 1991). The timetable of the onset of thyroid morphogenesis and the relation between the thyroid anlage and vessels of embryos during chick embryonic development are shown in Figure 1.

At the specification stage HH14 (50-53 hours

of incubation), the median thyroid anlage starts to be specified as a thickened epithelium in the midline of the anterior pharyngeal floor and appears to come into contact with the aortic sac (Fig. 1A, F). This thickening is located in the region between the first and second PAs. The thyroid anlage at the budding stage HH18 (65-69 hours) forms the diverticulum consisting of endoderm cells and invaginates into the mesenchyme, in close association with the aortic sac (Fig.1B, F). A 3D image shows almost the same situation between the specification and budding stages. Then the thyroid diverticulum buds off from the pharyngeal floor, migrates to the third PA arteries and elongates along them at the descent stage HH20 (70-72 hours) (Fig. 1C, G). The thyroid primordium bilobates along the route of the pair of the third PA arteries at the bilobation stage HH27 (4.5-5 days) (Fig. 1D, H). Finally, it matures and moves in close to and outside of the third PA arteries at the follicle formation stage HH38 (12 days) (Fig.1E, I).

Postotic CNC cells contribute to thyroid morphogenesis, but preotic CNC cells do not contribute directly to thyroid morphogenesis We examined the fate-mapping of postotic (r6–r8) (Fig. 2A-K) and preotic (mes– r5) (Fig.2L-V) CNC cells to thyroid morphogenesis using the quail-to-chick chimera technique, and confirmed their distribution patterns by 3D imaging. In postotic CNC chimeras, QCPN-positive quail cells migrated into the third and fourth PAs, although they were not seen around the thyroid anlage at the descent stage HH24.

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This was seen in 2 of 2 surviving chimeric embryos (Fig. 2B-E). At the bilobation stage HH35, postoic QCPN-positive CNC cells provided mesenchymal cells to the thyroid lobes (Fig. 2F-H), and contributed to formation of the UBs (Fig. 2I-K) (2 of 2 chimeric embryos). Preotic CNC cells migrated to the first and second PAs in the whole chimeric embryos (Fig. 2M-O). They migrated to the intercalated region between the thyroid placode and aortic sac, and on the sides of the placode at the late specification stage HH17. This was seen in 4 of 4 chimeric embryos. At the descent stage HH21, preotic CNC cells heavily surrounded the thyroid anlage and occupied the second PAs (3 of 8 chimeric embryos; Fig. 2P-R). At the later stages HH27 (4 of 4 chimeric embryos) and HH29 (2 of 2 chimeric embryos), however, almost none of the preotic CNC cells were found around the thyroid lobes (Fig. 2S-V), although many CNC cells occupied the first and second PAs. Therefore, preotic CNC cells are not considered to contribute directly to thyroid formation during development.

Both postotic and preotic CNC ablation cause thyroid bilobation defects at the bilobation stage HH31 Because postotic CNC ablation has been reported to cause thyroid bilobation defects at the follicle formation stage (14-16 days) (Bockman and Kirby, 1984), we first analyzed the effect of postotic (r6-r8) CNC ablation at earlier stages. Postotic CNC-ablated embryos (n=4) showed no abnormalities in the formation of the thyroid anlage at the descent stage HH23-24, in terms of either shape or volume, compared with controls (n=4) (Fig. 3A-C). By contrast, the defects became obvious at the bilobation stage HH31 (n=9, 100%), and included the complete absence of lobes (n=4, 45%), a single lobe located in the lateral side (n=3, 33%) or middle side (n=1, 11%), and unequal bilateral lobes (n=1, 11%) (Fig. 3D-F, Supplementary Table S2A). Sham-operated (n=6) and control (n=10) embryos showed no anomalies of the thyroid, UB’s or parathyroid. We also examined whether the CNC at the level of somites 4 to 5 (posterior to r6-8) contributed to thyroid bilobation, since a previous report described that CNC ablation in the regions comprising somites 1 to 5 had caused thyroid bilobation defects (Bockman and Kirby, 1984). The CNC ablation at the level of somites 4 to 5 caused no thyroid defects (n=3) (Fig. 3G), indicating that the CNC in this region did not make a major contribution to the thyroid bilobation. Taken together, these results showed that postotic CNC appeared to be involved in

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thyroid morphogenesis at the bilobation.

Moreover, the UBs (5 of 9 ablation

embryos, 56%) and parathyroid (6 of 9 ablation embryos, 67%) in postotic CNC-ablated embryos showed various defects with incomplete penetrance (Supplementary Table S2A). Although preotic CNC cells surrounded the thyroid anlage at the descent stage HH21, preotic CNC ablation (n=5) induced no anomalies in either the shape or volume of the thyroid anlage compared with controls (n=5) at the stages HH23-24 (Fig. 4A-C). We next examined the thyroid at the later bilobation stage HH31. At this stage, the preotic CNC ablation (n=13) resulted in normal thyroid bilobation (n=2, 15%) and thyroid bilobation defects (n=11, 85%). The defects were a single lobe (n=10) and unequal bilateral lobes (n=1) (Fig. 4D-G, Supplementary Table S2B). There was no significant difference in the volume of the thyroid anlage between the preotic CNC ablation embryos with thyroid defects (11 of 13 preotic CNC-ablated embryos) and controls (n=12) (Fig.4H), in contrast to the clear effect of postotic CNC cells on the volume of the thyroid anlage (Bockman and Kirby, 1984).

The

parathyroid and UBs were almost normal in preotic CNC-ablated embryos (Supplementary Table S2B). These findings suggested that the postotic and preotic CNC might play cooperative roles in thyroid bilobation. Since preotic CNC cells did not differentiate into thyroid tissue, they might influence thyroid bilobation indirectly.

Preotic CNC cells do not affect vessel formations  Postotic CNC (r6-r8) cells give rise to smooth muscle cells of the posterior PA arteries, which are the third, fourth, and sixth PA arteries, and the ablation of the CNC shows anomalies of the PA arteries (Bockman et al., 1987; Etchevers et al., 2001; Kulesa et al., 2000; Le Lievre and Le Douarin, 1975; Nishibatake et al., 1987; Waldo et al., 1996). On the other hand, preotic CNC (mes-r5) cells have been shown to contribute to the anterior PAs, third PAs, and associated structures (Etchevers et al., 2001; Kontges and Lumsden, 1996; Waldo et al., 1996). We performed PA artery angiography in preotic CNC-ablated embryos using an ink injection technique. At the descent stage HH23, the preotic CNC-ablated embryos (n=4) had the third, fourth and sixth PA arteries smaller than those of the control embryos (n=4), whereas the second PA arteries were enlarged and had not degenerated yet (Fig. 5A-D). Quail-chick chimera experiments revealed that preotic

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CNC cells hardly differentiated into smooth muscle cells of the third and fourth PA arteries, although some quail cells were found around the PA arteries (2 of 4 chimeric embryos) (Fig. 5E). At the follicle formation stage HH39, the control embryos (n=3) had the thyroid lobes that were placed outside and along the common carotid (third PA) arteries, while the preotic CNC-ablated embryos (n=2) showed normal carotid arteries with a unilateral and ectopic thyroid lobe that was placed inside of the left common carotid artery (Fig. 5F, G).

Therefore, although preotic

CNC cells may not directly contribute to the third PA arteries, they might indirectly affect their developmental processes.

The effects of preotic CNC cells on the migration pattern of postotic CNC cells at the descent stage HH24  Although the preotic CNC cells did not show a major contribution to smooth muscle cells in the posterior PA arteries, the size of the PA arteries was aberrant in the ablation embryos at the descent stage HH23. We therefore explored the idea that preotic CNC cells might influence the migratory pattern of postotic CNC cells. CM-DiI was used as a lineage tracer to label premigratory postotic CNC cells from 1st to 2nd -somites at 6-7 somite stages. In control embryos, DiI-labeled postotic CNC cells migrated to the third, fourth and sixth PA arteries at the descent stage HH24 (n=12) (Fig. 6A, B), and then they migrated to the heart at the bilobation stage HH27 (n=19) (Supplementary Fig. S1A) (Kirby, 1987; Miyagawa-Tomita et al., 1991). After ablating the preotic CNC, the labelled postotic CNC cells migrated to not only the third PAs, but also the second PAs (n=7) (Fig. 6C, D), and then they migrated to or around the mandibular arch at the bilobation stage HH27 (n=4) (Supplementary Fig. S1B). Moreover, we examined that after ablating from the preotic CNC to the postotic CNC in chick embryos, the quail NC from somites 1 to 2 or 3 was grafted to the chick embryo orthotopically as a partial rescue experiment (Fig. 6E-J). In the partially rescued chimeras, grafted postotic CNC cells were distributed in the second, third, and fourth PAs, and furthermore, they surrounded the thyroid diverticulum at the descent stage HH24 (7 of 7 rescued chimeras). Then we examined whether the re-routing of postotic CNC cells in the preotic-ablated embryos was due to cell proliferation or due to cell death in the thyroid primordia at the descent stage HH21, HH23-24 and the bilobation stage HH31. Positive cells for PCNA antibody were ubiquitously observed in the thyroid rudiments and surrounding mesenchyme. There was no difference in cell

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proliferation in the thyroid rudiments and surrounding mesenchyme between the postotic (n=9) and preotic CNC-ablated (n=9) embryos, similar to the findings in the controls (n=9) (data not shown). Some apoptotic cells were seen in the thyroid rudiments of the postotic (n=3) and preotic CNC-ablated (n=3) embryos as was also the case in the controls (n=3) at the descent stage HH21 (Supplementary Figure S2). Although there was no cell death in the thyroid rudiments of the postotic CNC-ablated, preotic CNC-ablated, and control embryos after stage HH21, more apoptotic cells appeared in the surrounding mesenchyme in the postotic (n=3) and preotic CNC-ablated (n=3) embryos than in the controls (n=3) at the descent stage 23-24.

At the late bilobation stage HH31, some apoptotic cells were seen in the

surrounding mesenchyme in the postotic CNC-ablated embryos (n=3), although apoptotic cells were not seen in the preotic-ablated (n=3) and control (n=3) embryos (data not shown) (Hirata and Hall, 2000).

Inactivation of Edn signaling causes thyroid bilobation defects Edn1, originally identified as a vasoconstrictor peptide, is one of the primary signals that establish the identity of CNC-derived tissues and organs in craniofacial and cardiovascular development through Ednra (Arima et al., 2012; Clouthier et al., 2010; Kempf et al., 1998; Kim et al., 2013; Kurihara et al., 1994; Kurihara et al., 1995; Ozeki et al., 2004). We have preliminarily reported that the thyroid is also affected in Edn1 knockout mice; however, this phenotype was not well characterized. To compare the effect of Edn1/Ednra inactivation and NC ablation on thyroid development, we analyzed abnormalities in thyroid development in chick embryos treated with bosentan and in Edn1 and Ednra knockout mice. At the bilobation stage HH37, bosentan-treated chick embryos (n=3) showed a unilateral thyroid lobe (n=2) (Fig. 7A, B) and ectopic lobes that were situated between the trachea and esophagus (n=1) (Fig. 7C). Control embryos demonstrated a normal thyroid bilobation that was located on both sides of the esophagus (n=3). At E18.5, wild-type (n=16) mice showed normal thyroid bilobation with the isthmus and fusion with the UBs, and they also showed the presence of follicular cells that were filled with PAS staining-positive colloids (Fig. 7D-G). In Edn1 knockout (n=7) and Ednra knockout (n=8) mice, a unilateral thyroid lobe was seen in three and five fetuses, respectively (Fig. 7H-M). The single thyroid lobe was separated from the UB in two Edn1 knockout and two Ednra knockout mice, and fused with the UB in one Edn1 knockout and three Ednra knockout mice.

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Discussion Several studies have reported that CNC cells contribute to thyroid morphogenesis. Studies using quail-chick chimeras with grafted quail mesencephalons and rhombencephalons have revealed that the CNC cells form connective tissue of the thyroid glands and C-cells of the UBs (Le Lièvre and Le Douarin, 1975; Polak et al., 1974). Ablation of the postotic CNC (over somites 1 to 5) results in thyroid gland anomalies that are missing on either both sides or one side, and they are reduced on either both sides or one side (Bockman and Kirby, 1984). However, it remains unclear which region of the cranial CNC contributes to thyroid morphogenesis. We therefore compared the distribution pattern and role of the postotic and preotic CNC in thyroid morphogenesis, especially bilobation, using quail-chick chimeras and CNC-ablated embryos. Our results showed that the postotic CNC contributes to thyroid formation directly, while the preotic CNC contributes to the bilobation process indirectly. Our findings showed that the preotic CNC, like the postotic CNC, plays an important role in thyroid development, and the two play cooperative roles in thyroid bilobation.

Thyroid bilobation defects The morphogenetic process of the chick thyroid gland was examined with respect to the five steps previously identified in three species (Fig. 1 and Supplementary Table 1).

At the specification stage to the descent stage HH24, postotic CNC cells

did not populate the region around the thyroid diverticulum. Moreover, postotic CNC ablation showed no effect on the thyroid anlage in terms of either shape or volume at the descent stage HH23-24.

After stage HH24, the postotic CNC cells

gave rise to connective tissue of the thyroid lobes and the postotic CNC-ablated embryos exhibited thyroid defects. Therefore, it was suggested that postotic CNC contributes directly to thyroid morphogenesis at the bilobation stage. On the other hand, preotic CNC cells encompassed the thyroid anlage at the late specification stage to the descent stage. Since it has been shown in Wnt1-Cre mice that NC cells surround the thyroid anlage at the late specification stage E9.5 (Jiang et al., 2000), these NC cells might be preotic CNC cells. At the late descent stage, preotic CNC ablation had no effect on the thyroid anlage in terms of shape and volume, and after this stage the preotic CNC cells were not close to the thyroid anlage and did not differentiate into cells of the thyroid gland, and preotic CNC ablation resulted in thyroid bilobation defects in which almost all of the embryos were missing one lobe.

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Preotic CNC cells may act on thyroid bilobation by surrounding the thyroid anlage from the specification stage to the descent stage. Therefore, it is suggested that preotic CNC cells contribute indirectly to thyroid morphogenesis at an earlier stage than the postotic CNC cells. In genetic studies in Shh, Hoxa3, Pax3, and Tbx1 knockout mice, as well as

Edn/Ednra knockout mice in the present study, a unilateral thyroid lobe was observed (Fagman et al., 2004; Fagman et al., 2007; Franz, 1989; Liao et al., 2004; Manley and Capecchi, 1995). Since these transcription factors and signals are involved in NC cell development, a deficiency in these factors might induce thyroid bilobation anomalies through NC cells. However, the mechanism underlying such an effect in knockout mice has not yet been elucidated.

Vessels are not related to thyroid bilobation In this study, preotic CNC-ablated embryos showed abnormal PA arteries at the descent stage. These abnormalities might suggest a temporal growth delay because preotic CNC ablation might cause a disturbance in the proliferation and migration of postotic CNC cells, which contribute to the development of smooth muscle cells of the PA arteries. At the later follicle formation stage, preotic CNC-ablated embryos showed the presence of normal common carotid arteries with a unilateral and ectopic thyroid lobe. Since the tunica media of the common carotid arteries are derived from r6 to r8 CNC cells and all arteries of the face and jaw, which branch off from the common carotid arteries are derived from the posterior diencephalon to r5 CNC cells (Etchevers et al., 2001), the postotic CNC might compensate for the damage to the preotic CNC-ablated embryos in forming normal common carotid arteries. Labeled postotic CNC in preotic CNC-ablated embryos and partial rescue in the chimeras showed that postotic CNC cells migrated to not only the third PA arteries, but also the second PA arteries at the descent stage, although postotic CNC cells might not be able to sufficiently maintain the normal growth of the PA arteries. Although rhombencephalic neural tube is capable of regenerating CNC cells (Couly et al., 1996; Sechrist et al., 1995; Suzuki and Kirby, 1997), our results are similar to a previous study (Kulesa et al., 2000) in chicks, in which the ablation of r5-r6 CNC allowed neighboring r7 CNC cells to reroute their trajectory to fill in the second PA. Rhombencephalic CNC cells have the capacity to reroute their migratory pathways and compensate for missing CNC cells after ablation of the neighboring population because the ablation causes an alteration of cell-cell interactions. On the other hand,

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it has also been reported in chicks that ablation of the postotic CNC appears not to induce regeneration or rerouting of the preotic CNC cells (Suzuki and Kirby, 1997). Thus the ablation studies of preotic and postotic CNC might indicate that the removal of these respective cell types induces different effects from the surrounding mesenchyme.

Clearly further studies will be needed, since the migration patterns

of postotic and preotic CNC cells might be interrelated. We examined cell proliferation and cell death in the thyroid rudiments of postotic and preotic CNC-ablated embryos. We found a high level of cell proliferation in the thyroid and surrounding mesenchyme in the postotic and preotic CNC-ablated embryos similar to those in controls at the descent stage, as previously reported in BrdU-uptake study, although the thyroid anlage at the specification and budding stage E9.5-E10.5 in mice is devoid of BrdU-labeled cells (Fagman et al., 2006). The results of apoptotic cell death in postotic and preotic CNC-ablated embryos were similar to those in controls at the descent stage.

Further study of this phenomenon

might also be warranted since both the postotic and preotic CNC-ablated embryos showed thyroid defects. While both Edn1 and Ednra knockout mice showed a unilateral thyroid lobe at E18.5, they showed the delay of the cranial extension, but no degeneration of the third PA arteries at the descent stage E12.5 (H. Kurihara, personal communication; Kim et al., 2013; Kurihara et al., 1995; Yanagisawa et al., 1998). Although Pax3 knockout mice are known to have heart defects such as PTA, the third PA arteries are seen at the descent stage E10.5 (Bradshaw et al., 2009). Therefore, the cranial extension of the third PA arteries from the descent stage to the bilobation stage may induce the thyroid anlage to the final position. In clinical reports, infants with congenital hypothyroidism have been reported to have a high frequency of other congenital anomalies, with congenital heart disease being the most frequent malformation (Chen et al., 2013; Olivieri et al., 2002). One of the causes is considered to be a disturbance of the proliferation and migration of CNC cells. Patients with 22q11 deletion syndrome demonstrate statistically significantly lower carotid artery bifurcations and a higher proportion of thyroid abnormalities based on computed tomography (de Almeida et al., 2009). In addition, no cardiovascular abnormalities have been reported in two cases of thyroid hemiagenesis (Korpal-Szczyrska et al., 2008). Therefore, the third PA arteries might be related to the final thyroid position, rather than to bilobation.

The ultimobranchial bodies and parathyroids

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The ultimobranchial bodies and parathyroid glands are derived from the endodermal epithelium of the pharyngeal pouches. The UBs develop from the fourth (mice) or sixth (birds) pharyngeal pouches and migrate to the thyroid lobes. There they give rise to C cells, which synthesize and secrete calcitonin, a serum calcium-lowering hormone. In most mammals, the UBs fuse with the thyroids, while in birds and lower vertebrates the UBs remain distinct. In birds, it has been reported that the cellular component of the UBs originates from the neural crest, whereas in mice the UBs consist of endothelium (Le Lièvre and Le Douarin, 1975; Polak et al., 1974; Kameda et al., 2007). Polak et al. have reported that CNC cells are the main cellular component of the UBs using quail-chick chimeras that is grafted from the mesencephalon to rhombencephalon (the caudal border of somite 6) of quail whole neural tube. Our study showed that QCPN-positive CNC cells were distributed to a part of the UBs bodies in quail-chick chimeras with postotic neural folds. This discrepancy might be due to the grafted CNC length, area, and whole neural tube or neural folds (Waldo et al., 1998). The parathyroids are derived from the third (mice), or third and fourth (birds) pharyngeal pouches and descend to the thyroid glands. The parathyroid gland produces parathormone, which controls the distribution of calcium in the body (Neves et al., 2012). Ablation of the postotic CNC induced UBs and parathyroid defects, while ablation of the preotic CNC showed no defect in the UBs and parathyroids. Therefore the postotic CNC seems to contribute to formation of the UBs and parathyroid glands, but the preotic CNC does not seem to contribute. Our results clarified the role played by the CNC in thyroid morphogenesis. Postotic CNC cells directly differentiate into thyroid mesenchymal cells, whereas preotic CNC cells appear to specifically, but indirectly contribute to thyroid bilobation.

Preotic CNC cells that did not contribute to thyroid formation induced

thyroid defects when they were removed. We could not find any explanation for this result in the present study and a search of the literature on cell proliferation and cell death.

Studies using partial rescued chimeras revealed that postotic CNC

cells surrounded the thyroid diverticulum, which showed rerouting of the postotic CNC cells, although we did not determine whether they populated connective tissues of the thyroid at later stages. We consider that the preotic CNC encompasses the thyroid anlage for thyroid bilobation and might induce thyroid bilobation through certain signals, such as Edn and Pax3, at an early stage. The postotic and preotic CNC might play different roles in thyroid bilobation, although postotic and preotic CNC ablation were associated with thyroid defects in our experiments. In

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other words, the mechanism by which bilobation is induced may be different from that by which the anlage is guided to the final position in thyroid morphogenesis, since the third PA arteries might act as guiding tracks for the thyroid to the final localization. However, further evidence is needed before any definitive conclusions can be made. Although we could not identify the signaling associated with the thyroid bilobation in this study, the preotic and postotic CNC were shown to contribute to thyroid morphogenesis indirectly and directly, respectively. Our results suggest that the prevalence of many congenital malformations, including the malformations of the cardiovascular system in infants with congenital hypothyroidism, is high and these findings should help to identify the causes of those congenital malformations.

Acknowledgements We thank Hiroaki Nagao for the histological analyses performed at Tokyo Women’s Medical University. We also thank Dr. Taro Kitazawa for the bosentan analysis, and all members of Dr. Kurihara’s laboratory at the University of Tokyo. We are particularly grateful to Actelion Ltd. for the gift of bosentan. This work was supported by a JSPS KAKENHI Grant (No. 25462147 to S M-T).

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Figure legends Figure 1. Timetable of the onset of thyroid morphogenesis and relationship between the thyroid anlage and vessels of embryos during chick development. (A-C) Sagittal sections

immunostained with Nkx2.1 antibody from the

specification to descent stages. (D, E) HE-stained transverse sections at the bilobation and follicle formation stages. (F-I) 3D images from the specification to follicle formation stages show the positional relationship between the thyroid anlage (magenta), vessels (blue) and outflow tract (oft, green). (A, F) The thyroid anlage (th) appears to come in contact with the aortic sac (as) at the specification stage HH14 (50-53 hours). (B, F) The thyroid anlage at the budding stage HH18 invaginates into the mesenchyme, in close association with the aortic sac. A 3D image shows almost the same situation between the specification (A) and budding (B) stages (65-69 hours). (C, G) The thyroid diverticulum buds off from the pharyngeal floor, migrates to the third PA arteries and elongates along them at the descent stage HH20 (70-72 hours). (D, H) The thyroid primordium bilobates along the route of the pair of the third PA arteries at the bilobation stage HH27 (4.5-5 days). The section and 3D image were taken at the late bilobation stage HH31. (E, I) Finally, the thyroid primordium matures and moves in close to the third PA arteries at the follicle formation stage HH38 (12 days). e, esophagus; f, follicle; t, trachea; pt, parathyroid; R (L) 1a, right (left) first PA artery; R (L) 2a, right (left) second PA artery; R (left) 3a, right (left) third PA artery; R4a, right fourth pharyngeal arch artery; R6a, right sixth pharyngeal arch artery. The scale bars represent 50 μm (A, E) and 100 μm (B-D).

Figure 2. Fate-mapping of postotic and preotic CNC cells in quail-chick chimeras. (A-K) Postotic CNC quail-chick chimeras grafted from rhombomeres (r)6 to r8, and (L-V) preotic CNC chimeras grafted from the mesencephalon to r5. (A, L) Schematic illustrations of postotic and preotic CNC chimeras. (B-E) Serial transverse sections immunostained with QCPN (brown, arrow head) (B-D) and a 3D image of the left-side view (E) of postotic CNC chimeras at the descent stage HH24. QCPN-positive postotic quail cells (brown in B-D, yellow in E) migrated into the third (3a) and fourth pharyngeal arch artery (4a) (blue), although they were not seen around the thyroid anlage (th, magenta) in the second pharyngeal arch artery (2a). (F-K) Sagittal sections of double immunostaining for QCPN (green) (F, I) and Nkx2.1 (red) (G, J) and their merged image (H, K) of the thyroid rudiment (F-H) and

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ultimobranchial body (ub) (I-K) in postotic CNC chimeras at HH35. Nkx2.1 was expressed in the parenchymal cells of the thyroid lobes and the QCPN-positive CNC cells indicate mesenchymal cells. In the ultimobranchial body, Nkx2.1 was expressed in the center of parenchymal cells and the QCPN-positive CNC cells were distributed to around the center of the body. (M-O) Whole-mount (M), sagittal sections immunostained with QCPN (N), and dorsal aspect of the reconstructed 3D image (O) in preotic CNC chimeras at the late specification stage HH17. The preotic CNC cells were mainly distributed in the 1st and 2nd pharyngeal arches. The QCPN-positive CNC cells were scattered at the front and sides of the thyroid placode. (P-R) Whole-mount (P), transverse section stained with QCPN (Q), and the dorsal aspect of the 3D image (R) at the descent stage HH21. Preotic CNC cells were observed in the maxillary (mx) and mandibular (md) processes and encompassed the thyroid anlage. (S-V) Whole-mount (S, U) and sagittal (T) and transverse section (V) stained with QCPN at the bilobation stages, HH27 and 29. Although preotic CNC cells were seen in the maxillary and mandibular processes, preotic CNC cells were not seen around the thyroid anlage at this stage. oft, outflow tract (green) ; R (L) 2a, right (left) second pharyngeal arch artery; R (L) 3a, right (left) third pharyngeal arch artery. The scale bars present 500 μm (P, S-V), 200 μm (B-D, M, Q), and 50 μm (F-K, N). Figure 3. Effect of postotic CNC ablation on the thyroid anlage at the descent stage HH23-24 and bilobation stage HH31 chick embryos. (A-C) Transverse sections with HE staining (A, B), the 3D images (A’, B’) and the volume of the 3D images (C) at the descent stage HH23-24. Postotic CNC ablation (postCNC-ab) showed no effect on the thyroid anlage (th, magenta) in terms of either shape or volume at the descent stage HH23-24. (D-G) Transverse sections with HE staining at the bilobation stage HH31 (D-G). The control (cont, D) and sham-operated (sham, E) embryos showed normal thyroid bilobation. Postotic CNC-ablated embryos showed a single thyroid lobe (F), and CNC ablation from somites 4 to 5 embryos showed normal thyroid bilobation (sm4-5 ab, G). e, esophagus; pt, parathyroid; t, trachea. The scale bars represent 200 μm. Figure 4. Effect of preotic CNC ablation on the thyroid anlage at the descent stage HH23-24 and bilobation stage HH31 chick embryos. Transverse sections with HE staining (A, B), the 3D images (A’, B’, D-G) and the volume of 3D images (C, H) in preotic CNC-ablated embryos (preCNC-ab) at the

26

descent stage HH23-24 and bilobation stage HH31. Control (cont) (A, A’, D), sham-operated (sham) (E) and preotic CNC-ablated (B, B’, F, G) embryos are shown. (A-C) Preotic CNC-ablated embryos showed no effect on the thyroid anlage in terms of shape or volume compared with control embryos at the descent stage HH23-24 (C, P>0.1). (D-H) There was no significant difference in the volume of the thyroid anlage between the preotic CNC ablation embryos with thyroid defects and controls at the bilobation stage HH31 (H, P>0.1). Preotic CNC-ablated embryos caused thyroid bilobation defects, including a unilateral lobe (F) and unequal bilateral lobes (G).

th, thyroid. The scale bars represent 200 μm.

Figure 5. Effect of preotic CNC ablation on the pharyngeal arch arteries. (A-D) Angiography by ink injection in control (cont) (A, B) and preotic CNC-ablated (preCNC-ab) (C, D) embryos at the descent stage HH23. The third (3a), fourth (4a) and sixth (6a) pharyngeal arch arteries in the preotic CNC-ablated embryos were smaller than those in the control embryos, and the second (2a) pharyngeal arch arteries increased in size. (E) Immunostaining for QCPN antibody showed that the preotic CNC cells were partially scattered around the third (R3a) and fourth (R4a) pharyngeal arch arteries in the preotic CNC-grafted quail-chick chimera at HH23. Transverse section. (F) The control embryos at HH39 showed normal thyroid bilobation and final localization. The thyroid glands (th) locate above the subclavian arteries and outside of the common carotid arteries. (G) The preotic CNC-ablated embryos at HH39 showed a unilateral and ectopic thyroid lobe (n=2). The thyroid gland was located inside of the common carotid arteries at a lower position than those in the control. e, esophagus; Lcc, left common carotid artery; L (R) sc, left (right) subclavian artery; oft, outflow tract; t, trachea. The scale bars represent 200 μm (A-E) and 500 μm (F, G).

Figure 6. Preotic CNC cells have an effect on the migration of postotic CNC cells at the descent stage HH24 chick embryos. (A-D) Tracking of postotic CNC cells by DiI labelling. CM-DiI was injected into the postotic CNC (somites 1-2) of the control embryos (cont) (A). Control embryos show that the labeled postotic CNC cells migrated to the third (3), fourth (4) and sixth (6) pharyngeal arches at the descent stage (B). (C, D) After the ablation of preotic CNC (preCNC-ab) embryos, the labeled postotic CNC cells migrated not only to the third pharyngeal arch but also the second (2) pharyngeal arch. Dorsal views at 6-7 somite

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stages (arrowheads indicate dye location in A, C). Right lateral views at HH24 (B, D). (E-J) The labeled quail postotic CNC (somites 1-2) were transplanted to the CNC over preotic to postotic-ablated chick embryos orthotopically (arrowheads indicate dye-labeled quail CNC in E). A whole-mount (F) and sagittal section (G) stained with QCPN antibody at descent stage HH24 are shown. The results of double immunostaining for QCPN (green) and Nkx2.1 (red) in sagittal sections (H, I), and their merged image in the thyroid (th) area (J) are also presented. In partial rescued quail-chick chimeras, postotic CNC cells were distributed in the second (2a), third (3a), fourth and sixth pharyngeal arches and surrounded the thyroid rudiment. oft, outflow tract. The scale bars present 200 μm (B, D, F) and 50 μm (G-J). Figure 7. Thyroid bilobation defects in bosentan-treated chick embryos at the bilobation stage HH37 and Edn1 and Ednra knockout mice at E18.5. (A-C) Control (cont) chick embryos showed normal thyroid (th) bilobation that was located on both sides of the esophagus (e) (A). Bosentan-treated chick embryos showed a unilateral (arrowhead in B) and ectopic thyroid lobes that were located between the esophagus and trachea (t) (arrowheads in C) at the bilobation stage HH37. Transverse sections stained with Nkx2.1 antibody (A, B) and HE (C) are shown. (D-M) The results in wild-type mice showed that thyroid bilobation containing the isthmus (is) (D) fuses with the ultimobranchial body (th-ub) (E-G) and forms PAS staining-positive colloid-filled follicles (arrowhead in F, G, J, M) at E18.5. Edn1 (H-J) and Ednra (K-M) knockout mice showed a unilateral thyroid lobe. A single thyroid separated from the ultimobranchial body (ub) (J) or fused to the ultimobranchial body (M) was seen in Edn1 and Ednra knockout mice. In spite of being separated from the ultimobranchial body, the unilateral thyroid lobe formed follicular cells filled with PAS-positive follicles. The results of HE staining are shown in (D). Transverse sections with immunostaining for calcitonin are shown in brown and PAS staining is shown with a deep red-purple color (E-M). F, G, I, J, L, M are magnifications of the right- and left-side views of E, H and K. The scale bars represent 500 μm (A-C), 200 μm (D, E, H, K) and 50 μm (F, G, I, J, L. M).

Supplementary data Supplementary Table S1. Timetable of the onset of morphogenetic events during thyroid development in four

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species.

Supplementary Table S2. (A) Effects on the thyroid, ultimobranchial body and parathyroid in postotic CNC-ablated embryos at HH31. PTA, persistent truncus arteriosus. (B) Effects on the thyroid, ultimobranchial body and parathyroid in preotic CNC-ablated embryos at HH31.

Supplementary Figure S1. Preotic CNC cells have an effect on the migration of postotic CNC cells at the bilobation stage HH27. CM-DiI was injected into the postotic CNC (somites 1-2) of the control (cont, A) and preotic CNC-ablated embryos (preCNC-ab, B). Although postotic CNC cells (arrowheads in A) migrated to the heart in control embryos at the bilobation stage HH 27, they migrated to or around the mandibular

processes (md) in preotic

CNC-ablated embryos (arrowhead in B). The scale bars present 200 μm.

Supplementary Figure S2. TUNEL analysis of the thyroid rudiment in postotic CNC and preotic CNC-ablated chick embryos. TUNEL analysis at the descent stage HH21 (A-C), late descent stage HH23-24 (D-F), and late bilobation stage HH31 (G-I). Results are shown for the control chick embryos (cont) (A, D, G), postotic CNC-ablated embryos (postCNC-ab) (B, E, H), and preotic CNC-ablated embryos (preCNC-ab) (C, F, I). At the descent stage HH21, some apoptotic (TUNEL-positive) cells were seen in the thyroid anlage (th), endoderm (e), and surrounding mesenchyme of the control (A), postotic CNC-ablated (B), and preotic CNC-ablated (C) embryos. No apoptotic cells were seen in the thyroid anlage of the control (D, G), postotic CNC-ablated (E, H), and preotic CNC-ablated embryos (F, I) after the descent stage HH21, although many apoptotic cells were seen in the pharyngeal mesenchyme of the postotic and preotic CNC-ablated embryos compared with those in the controls at the late descent stage HH23-24. The scale bar represents 50 μm.

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Highlights Ablation of either postotic or preotic neural crest leads to thyroid malformation. Thyroid bilobation is associated with both postotic and preotic neural crest cells. Postotic and preotic neural crest differently contribute to thyroid development.

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R3a

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