Immunohistochemical profiling of the ultimobranchial remnants in the rat postnatal thyroid gland

For Peer Review IMMUNOHISTOCHEMICAL PR OFILING OF THE ULTIMOBRANCHIAL REMNANTS IN THE RAT POSTNATAL THYROID GLAND Journal: Journal of Morphology Manuscript ID Draft Wiley - Manuscript type: Research Article Date Submitted by the Author: n/a Complete List of Authors: Vázquez-Román, Victoria; Medicine School. University of Seville. , Cytology and Histology Utrilla, José; Medicine School. University of Seville, Citology and Histology Fernández-Santos, José; Medicine School. University of Seville, Citology and Histology Martín-Lacave, Inés; Medicine School. University of Seville, Cytology and Histology Keywords: ultimobranchial follicle (UBF), immunohistochemistry (IHC), rat thyroid John Wiley & Sons Journal of Morphology For Peer Review Vázquez Román et al. 1 IMMUNOHISTOCHEMICAL PROFILING OF THE ULTIMOBRANCHIAL 1 REMNANTS IN THE RAT POSTNATAL THYROID GLAND 2 Victoria Vázquez-Román*, José C. Utrilla*, José M. Fernández-Santos and Inés 3 Martín-Lacave. 4 5 Department of Normal and Pathological Cytology and Histology, School of Medicine, 6 University of Seville, Spain. 7 8 Short Title: Immunohistochemistry of ultimobranchial remnants in rat thyroid 9 10 * Victoria Vázquez-Román and José C. Utrilla contributed equally to this work. 11 12 13 Corresponding author: 14 Inés Martín-Lacave 15 Dpt. Normal and Pathological Cytology and Histology 16 School of Medicine 17 Avda. Sánchez-Pizjuán s/n 18 41009 Seville, Spain. 19 Email: [email protected] 20 21 Page 1 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 2 ABSTRACT 22 23 Ultimobranchial (UB) remnants are a constant presence in the thyroid throughout rat 24 postnatal life; however, the difficulty in identifying the most immature forms from the 25 surrounding thyroid tissue prompted us to search for a specific marker. With that 26 objective, we applied a panel of antibodies reported to be specific for their human 27 counterpart, solid cell nests (SCNs), using double immunohistochemistry and 28 immunofluorescence. Our results demonstrated that cytokeratin 34βE12 and p63 are 29 highly sensitive markers for the immunohistologic screening of UB remnants, 30 independently of their maturity or size. Furthermore, rat UB follicles (UBFs) coincided 31 with human SCNs in the immunohistochemical pattern exhibited by both antigens. In 32 contrast, the pattern displayed for calcitonin and thyroglobulin differs considerably but 33 is compatible with the hypothesis that UB cells can differentiate into both types of 34 thyroid endocrine cells. This hypothesis agrees with recent findings that thyroid C cells 35 share an endodermic origin with follicular cells in rodents. We suggest that the 36 persistence of p63-positive undifferentiated cells in UB remnants may constitute a 37 reservoir of basal/stem cells that persist beyond embryogenesis from which, in certain 38 unknown conditions, differentiated thyroid cells or even unusual tumours may arise. 39 40 41 42 43 44 Keywords: ultimobranchial follicle (UBF), immunohistochemistry (IHC), rat thyroid. 45 46 Page 2 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 3 INTRODUCTION 47 In mammals, the thyroid gland consists of two endocrine cell populations, follicular 48 cells and C cells, with different functions and embryonic origins. During development, 49 the thyroid diverticulum, which is derived from the ventral pharyngeal floor, moves 50 caudally along the midline and forms two lateral lobes, thus giving rise to follicular 51 cells. In parallel, the two UB bodies (UBBs) are separated from the last pair of 52 pharyngeal pouches and approach the thyroid vesicle until their fusion, becoming 53 embedded in the thyroid lobes and giving rise to C cells (Fagman and Nilsson, 2011; 54 Westerlund et al., 2008). In contrast, in lower vertebrates, such as birds and fishes, no 55 fusion of the UBBs with the thyroid lobes occurs, but they remain as separate glands 56 called UB glands; hence, the thyroid gland is exclusively composed of follicular cells 57 (Fagman and Nilsson, 2010). 58 The UBBs, apart from forming C cells, remain in the adult thyroid gland as rather 59 complex structures considered as embryonic remnants with unknown significance. 60 These structures show differences among species and have been described in the 61 literature under different names. Specifically, they have been extensively studied in rats, 62 where they are mostly known as "ultimobranchial follicles" (UBFs) (Martin-Lacave et 63 al., 1992; Rao-Rupanagudi et al., 1992; Van Dyke, 1944; Wollman and Neve, 1971a; b) 64 and in humans, where they are termed "solid cell nests” (SCNs) (Beckner et al., 1990; 65 Harach, 1988; Harach et al., 1993). 66 It is generally accepted that both UBFs in rats and SCNs in humans are embryonic 67 remnants of UBBs and, therefore, share a common origin (Bellevicine et al., 2012; 68 Wollman and Hilfer, 1977; 1978). Nevertheless, despite this common embryonic origin, 69 there are differences regarding the appearance and evolution of these UB remnants 70 Page 3 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 4 during postnatal life in both species. Specifically, in rats, these structures appear in all 71 the thyroid glands, although they adopt different morphological patterns throughout 72 postnatal life (Vazquez-Roman et al., 2013). Thus, in young rats (0-180 days), they 73 evolve from narrow cellular nests to tubular forms, the so-called "immature UBFs”. In 74 addition, “mixed follicles”, which are partially formed by UBFs fusing to usual thyroid 75 follicles, may also be observed in young rats. In contrast, in adult rats (6-15 months) 76 and old rats (18-24 months), mature cystic forms predominate, the so-called "mature 77 UBFs", which are rather rounded, with stratified flattened cells in the wall, and cellular 78 debris in the lumen. Moreover, in adult and old rats, an unusual progression of the 79 forms described above can be found that resembles enormous onion-like structures that 80 we have termed “UB cystoadenomata”. With the exception of UB cystoadenomata, 81 there are difficulties in the microscopic identification of UB remnants from the 82 surrounding parenchyma due to either relative small size or their uncharacteristic form 83 (Vazquez-Roman et al., 2013). 84 In humans, diverse immunohistochemical (IHC) studies have been performed to 85 characterize the cellular composition of SCNs (Burstein et al., 2004; Cameselle-Teijeiro 86 et al., 1994; Mizukami et al., 1994; Preto et al., 2004; Reis-Filho et al., 2003; Rios 87 Moreno et al., 2011). According to those studies, two cell types form the SCNs, which 88 are referred to as “main cells” and “C cells”. Specifically, “main cells” can be 89 immunostained for some cytokeratins (CK34βE12, CK7, CK11, CK19, 90 carcinoembryonic antigen (CEA), galectina-3, as well as different markers expressed in 91 the basal/stem cells of stratified epithelium, such as p63, bcl-2 or telomerase. In 92 contrast, C cells are positive for calcitonin (CT), calcitonin gene-related peptide 93 Page 4 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 5 (CGRP), chromogranin and thyroid transcription factor (TTF-1), but lack 94 immunoreactivity for p63. 95 In rats, no IHC studies on UBFs have been reported, with the exception of two articles 96 published by us in which the occasional presence of CT and thyroglobulin (Tg) in the 97 wall was analysed (Conde et al., 1992; Vazquez-Roman et al., 2013). No published data 98 describe a precise staining method to specifically identify UBFs in the rat independently 99 of their morphological pattern and magnitude. In addition, there are no specific IHC 100 analyses that could help to clarify their cellular composition and whether the UBFs are 101 homogeneous structures composed only of “U-cells”, as stated by Wollman and Neve 102 (Wollman and Neve, 1971a; b), or if they are rather heterogenic structures, such as 103 SCNs (Cameselle-Teijeiro et al., 1994; Harach, 1988; Martin et al., 2000). Furthermore, 104 no new evidence has been provided to clarify whether UB remnants contribute to the 105 formation of C cells and thyroid follicular cells during postnatal life in mammals, and 106 their possible contribution to certain types of pathology at the thyroid level remains to 107 be elucidated. Therefore, the main objectives of the present article were addressed to 108 shed light on the aforementioned aspects. 109 110 111 Page 5 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 6 MATERIALS AND METHODS 112 Selection of samples 113 The study material consisted of sixty formalin-fixed, paraffin-embedded and serially 114 sectioned thyroid glands of Wistar rats, of both sexes and different ages, in which we 115 previously described the appearance of different morphological patterns that adopt the 116 UB remnants throughout rat postnatal life (Vazquez-Roman et al., 2013). Specifically, 117 25 cases of “immature UBFs” (detected in young rats), 25 cases of “mature UBFs” 118 (detected in adult rats), and 10 cases of UB cystadenomata (detected in old rats) were 119 analysed. All experiments were conducted in accordance with the guidelines proposed 120 in The Declaration of Helsinki (http://www.wma.net) involving the use of laboratory 121 animals. 122 Immunohistochemical Analysis 123 Single Immunohistochemistry 124 Once a particular UBF pattern was detected in a thyroid section, consecutive sections of 125 the same thyroid gland were selected to proceed with the IHC study. Silane-coated 126 sections were dewaxed in xylene and hydrated through graded alcohols. Next, an 127 antigen retrieval step using EnVision Flex Target Retrieval High pH (DAKO, Denmark) 128 was performed in a heating instrument, PTLink (DAKO), at 96ºC for 20 min, according 129 to the manufacturer’s instructions. The slides were immersed in a washing solution 130 (Wash Buffer DAKO) for 5 min. Then, the sections were treated with 3% hydrogen 131 peroxide to block endogenous peroxidase activity for 20 min. The slides were then 132 incubated with a panel of primary antibodies (see Table 1), at 4 °C overnight in a 133 humidified chamber. EnVision Flex/HRP (DAKO) was used as the labelling system 134 according to the manufacturer’s instructions, and 3,3’-diaminobenzidine 135 Page 6 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 7 tetrahydrochloride (DAB) solution (Sigma–Aldrich, Germany) was used as chromogen. 136 The slides were counterstained with haematoxylin, dehydrated and coverslipped. 137 Photomicrographs of the samples were performed using an Olympus photomicroscope 138 (Vanox AHBT3). 139 Double Immunohistochemistry 140 To analyse the colocalization of CT or Tg with p63, double IHC labelling was 141 performed with an antigen retrieval step in between the two staining sequences to 142 prevent cross-reactions among reagents, according to Lan et al. (Lan et al., 1995). 143 Briefly, the sections were dewaxed and pretreated in the same manner as described 144 above for single IHC but without the antigen retrieval step. In the first immunostaining 145 sequence, the specific antibody (anti-CT or anti-Tg) was incubated at 4 ºC overnight 146 and followed either by the LSAB/Alkaline Phosphatase system (DAKO), for CT, or 147 EnVision Flex, for Tg. The enzymatic reaction was visualized with Fast Red or DAB148 Cobalt-chloride (Sigma–Aldrich) as chromogens, respectively. Next, an antigen 149 retrieval step using EnVision Flex Target Retrieval High pH, as described above, was 150 intercalated before the second immunostaining sequence started. The second specific 151 antibody, anti-p63, was incubated at 4 ºC overnight, and EnVision Flex/HRP or 152 LSAB/Alkaline Phosphatase (DAKO) were used as the labelling systems. After colour 153 development with DAB or DAB-Cobalt-chloride as chromogens, the slides were 154 counterstained with haematoxylin and coverslipped in an aqueous permanent medium. 155 To analyse the colocalization of TTF-1 with CK34βE12, a similar technique was used 156 but before applying the first specific antibody (anti-TTF-1) an antigen retrieval step 157 with EDTA buffer, pH 9 (DAKO, Denmark) was carried out. The specific binding was 158 developed using streptavidin-biotin-peroxidase technique (LSAB+/HRP kit, DAKO), 159 Page 7 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 8 and DAB (Sigma–Aldrich, Germany) as chromogen. In the second immunostaining 160 sequence, the specific antibody (anti-CK34βE12) was incubated at 4 ºC overnight, 161 followed by the LSAB/Alkaline Phosphatase system (DAKO) and enzymatic 162 development with Fast-Red as chromogen. 163 Double Immunofluorescence (IF) 164 To analyse the colocalization of p63 or CK34βE12 with CT or Tg, respectively, by 165 double IF, thyroid sections were dewaxed, hydrated and pretreated for antigen retrieval 166 using Target Retrieval High pH buffer, as described above. Nonspecific binding was 167 blocked with 10% normal donkey serum for 15 min (Jackson ImmunoResearch 168 Laboratories). Then, the monoclonal primary antibody, either p63 or CK34βE12, was 169 added for 1 h at room temperature in a humidified chamber. Subsequently, the slides 170 were washed and incubated with Cy3-labeled donkey anti-mouse IgG secondary 171 antibody (1:100, Jackson ImmunoResearch Laboratories) for 30 min at room 172 temperature in a humidified chamber. After washing in PBS, the second 173 immunostaining sequence started. The slides were then incubated with a specific 174 polyclonal anti-CT antibody or anti-Tg antibody and, subsequently, with Cy2-labeled 175 donkey anti-rabbit IgG antibody (1:100, Jackson ImmunoResearch Laboratories) under 176 the same conditions as before. After washing in PBS, DAPI (Sigma-Aldrich) was added 177 for nuclear counterstaining, and the slides were coverslipped with antifading mounting 178 medium (Mowiol 4-88, Sigma-Aldrich). The sections were visualized with a 179 fluorescence microscope (Olympus BX50, Hamburg, Germany). Images were acquired 180 using an ORCA-03G digital camera (Hamamatsu, Bridgewater, Rockville, USA) and 181 analysed using Image PRO-PLUS 7.0 software (Media Cybernetics, USA). The antigen 182 combinations were as follows: p63-CT, p63-Tg, CK34βE12-CT, and CK34βE12-Tg. 183 Page 8 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 15 most peripheral cells, as it also occurs in different forms of SCNs (Reis-Filho et al. 302 2003; Burstein et al. 2004). In contrast, the patterns displayed for CT and Tg differed 303 considerably between both types of UB remnants. In rats, CT-positive cells were only 304 found in very scarce immature UBFs or in the walls of UBFs presenting aberrant 305 localization at the thyroid level, as we previously reported (Martin-Lacave et al. 1992). 306 Furthermore, no increase in the number of C cells was found in the background thyroid 307 tissue near the UB remnants. Therefore, we conclude that UBFs do not normally 308 contribute to the formation of new C cells during rat postnatal life, which is the opposite 309 to that observed in humans in which numerous C cells are intermingled with main cells 310 in SCNs (Cameselle-Teijeiro et al., 2005b; Harach and Wasenius, 1987; Mizukami et 311 al., 1994; Reis-Filho et al., 2003; Rios Moreno et al., 2011). Nevertheless, the capacity, 312 if any, of those postnatal C cells to migrate from UB remnants along the connective 313 tissue to occupy their definitive position in relation to thyroid follicles, likely through an 314 epithelial mesenchymal transition process (EMT) (Acloque et al., 2009), remain to be 315 elucidated. 316 317 Some differences were found between UBFs and SCNs in relation to their IHC pattern 318 for Tg. Specifically, Tg was observed at the cytoplasmic level in scarce p63-negative 319 cells as well as in some colloid-like drops that appeared within the wall of immature 320 UBFs. Conversely, in humans, no Tg-positive cells have ever been reported as forming 321 a part of SCNs (Autelitano et al., 1987; Cameselle-Teijeiro et al., 2005b; Cameselle322 Teijeiro et al., 1994; Mizukami et al., 1994; Reis-Filho et al., 2003; Rios Moreno et al., 323 2011). Accordingly, we also observed scarce cells expressing TTF-1, a common marker 324 of differentiated thyroid cells, either follicular cells (Lazzaro et al., 1991) or C cells 325 Page 15 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 16 (Suzuki et al., 1998), in the UBF wall. This finding is compatible with the hypothesis 326 that UB cells could differentiate into both endocrine cell populations, as we previously 327 suggested (Martin-Lacave et al., 1992; Moreno et al., 1989). This hypothesis agrees 328 with the findings of Kameda and cols. (Kameda et al., 2007) who discarded that 329 mammalian thyroid C cells are derived from the neural crest, as it occurs with avian CT330 producing cells of the UB gland (Kameda, 1995). The authors demonstrated by fate 331 mapping of neural crest cells in both Wnt1-Cre/R26R and Connexin (Cxn) 43-lacZ 332 transgenic mice that neural crest cells did not colonize the fourth pharyngeal pouch or 333 the UBB (Kameda, 2016; Kameda et al., 2007). Furthermore, Johansson et al. have 334 recently clarified using lineage tracing in Sox17-2A-iCre/R26R mice that pharyngeal 335 endoderm-derived cells give rise to C cells (Johansson et al., 2015). Both findings 336 together indicate that mouse thyroid C cells are derived from the endodermal epithelial 337 cells of the fourth pharyngeal pouch; hence, they share an endodermic origin with 338 follicular cells, which are derived from the ventral pharyngeal floor. Therefore, the 339 present data are compatible with the hypothesis that the foregut endoderm gives rise to 340 both thyroid endocrine cell types in the rat, as previously proposed by Westerlund et al. 341 for the mouse (Westerlund et al., 2008). 342 Based on the fact that all normal rat thyroid glands have UBFs throughout adult life, a 343 question arises about the meaning of the persistence of the UB remnants. The same 344 question was raised by Ozaki et al. for human SCNs (Ozaki et al., 2011). We agree with 345 those authors that the answer to this question is likely related to the constant presence of 346 undifferentiated cells associated with UB remnants in mammals, independently of their 347 own peculiarities. The existence of p63-positive cells suggests that they may constitute 348 a reservoir of basal/stem cells that persists beyond embryogenesis from which, in 349 Page 16 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 17 certain unknown conditions, differentiated thyroid cells or even unusual tumours, such 350 as rat UB cystoadenomata (Vazquez-Roman et al., 2013), human mucoepidermoid 351 (Cameselle-Teijeiro et al., 1994; Harach et al., 1993) or mixed medullary and follicular 352 carcinomas (Matias-Guiu, 1999), may arise. Moreover, Ozaki et al. (Ozaki et al., 2012) 353 recently demonstrated the appearance of numerous clear immature cells after partial 354 thyroidectomy that expressed keratin14 and Foxa2, the definitive endoderm lineage 355 marker; these cells could be derived from the UBB, suggesting a critical role for UB 356 remnants in thyroid regeneration (Okamoto et al., 2013). 357 There is evidence for the presence of adult stem cells of endodermic origin in the human 358 thyroid gland that express OCT4, a classical marker of stem cells (Thomas et al., 2006). 359 Stem cells, ranging from 0.3%-1.4% of the total cell population, were also obtained 360 from mouse thyroid glands; half of the cells expressed OCT4 in addition to other 361 specific stem cell markers, such as ABCG2 and nucleostatin (Hoshi et al., 2007). It has 362 repeatedly been proposed that SCNs may represent a pool of stem cells that could 363 contribute to the histogenesis of thyroid cells and thyroid regeneration in adult life 364 (Preto et al., 2004; Reis-Filho et al., 2003). According to our results, the same could be 365 suggested for UB remnants in the rat thyroid gland. Nevertheless, further studies are 366 required to understand the true nature of undifferentiated UB cells and their relationship 367 to differentiated thyroid cells throughout postembryonic life. 368 369 AUTHOR CONTRIBUTIONS 370 VVR: acquisition of data, data analysis and interpretation. JCU: modified the 371 methodology, acquisition of data, data analysis and interpretation. JFS: artwork, 372 Page 17 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 18 manuscript revisión and approval. IML: concept and design of the study, wrote the 373 manuscript. Neither author has any conflict of interest to declare. 374 375 376 ACKNOWLEDGEMENTS 377 This work was supported by grants from the Consejería de Innovación, Ciencia y 378 Empresa (refs. CTS-439/2011), and from the Consejería de Salud (ref. PI-0051-2013), 379 Junta de Andalucía, Spain. The authors thank Mr. John Brown for English language 380 corrections. 381 382 383 Page 18 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 19 REFERENCES 384 385 Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. 2009. Epithelial-mesenchymal 386 transitions: the importance of changing cell state in development and disease. J Clin 387 Invest 119(6):1438-1449. 388 Autelitano F, Santeusanio G, Di Tondo U, Costantino AM, Renda F, Autelitano M. 1987. 389 Immunohistochemical study of solid cell nests of the thyroid gland found from an 390 autopsy study. Cancer 59(3):477-483. 391 Beckner ME, Shultz JJ, Richardson T. 1990. Solid and cystic ultimobranchial body remnants in 392 the thyroid. 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Age-related changes in thyroid 478 lesions and function in F344/DuCrj rats. Exp Anim 44(1):57-62. 479 Thomas T, Nowka K, Lan L, Derwahl M. 2006. Expression of endoderm stem cell markers: 480 evidence for the presence of adult stem cells in human thyroid glands. Thyroid 481 16(6):537-544. 482 Van Dyke JH. 1944. Behavior of Ultimobranchial Tissue in the postnatal Thyroid Gland: The 483 origin of Thyroid Cistoadenomata in the rat. . Anat Rec 88:17. 484 Vazquez-Roman V, Utrilla JC, Fernandez-Santos JM, Conde E, Bernabe R, Sampedro C, Martin-485 Lacave I. 2013. Postnatal fate of the ultimobranchial remnants in the rat thyroid gland. 486 J Morphol 274(7):725-732. 487 Westerlund J, Andersson L, Carlsson T, Zoppoli P, Fagman H, Nilsson M. 2008. Expression of 488 Islet1 in thyroid development related to budding, migration, and fusion of primordia. 489 Dev Dyn 237(12):3820-3829. 490 Wollman SH, Hilfer SR. 1977. Embryologic origin of various epithelial cell types in the thyroid 491 gland of the rat. Anat Rec 189(3):467-478. 492 Wollman SH, Hilfer SR. 1978. Embryologic origin of the various epithelial cell types in the 493 second kind of thyroid follicle in the C3H mouse. Anat Rec 191(1):111-121. 494 Wollman SH, Neve P. 1971a. Postnatal development and properties of ultimobranchial follicles 495 in the rat thyroid. Anat Rec 171(2):247-258. 496 Wollman SH, Neve P. 1971b. Ultimobranchial follicles in the thyroid glands of rats and mice. 497 Recent Prog Horm Res 27:213-234. 498 499 500 501 Page 21 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 22 FIGURE LEGENDS 502 Figure 1. IHC profile of the different forms of UBFs appearing in rat postnatal life. A) 503 Immature forms; B) Mature cystic UBF; C) UB cystadenomata. CK34βE12 and p63 are 504 the most specific markers of UB remnants, which are clearly immunostained compared 505 to the surrounding normal thyroid follicles. In contrast, immunostaining for 506 CKAE1/AE3 or TTF-1 was also shared with the rest of the thyroid tissue. Bar: A1-B4, 507 C3-C4=50 µm, C1-C2 = 200 µm. 508 Figure 2. Double immunostaining for CT and p63 in different UBFs. P63 509 immunopositivity (in brown) is mainly confined to the nuclei of peripheral cells in both 510 immature (A) and mature (B) UBFs. Nevertheless, in immature forms, few scattered 511 CT-positive cells (in red, arrows) could also be observed intermingled with p63-positive 512 cells. Bar =50 µm. 513 Figure 3. Double immunostaining for Tg and p63 in immature UBFs. P63 514 immunopositivity (in dark blue) is located in most peripheral cells of the UBF according 515 to a nuclear pattern. In contrast, Tg (in brown) exhibited a rather heterogeneous pattern, 516 with scarce cells and colloid-like drops (arrow) that were immunopositive for Tg among 517 p63-positive, Tg-negative cells (A). In panel B, one mixed follicle immunostained for 518 Tg (arrow) could be observed merging from the UBF wall. Bar =25 µm. 519 Figure 4. Double IF for p63 and CT (A) and CK34βE12 and CT (B) in immature UBFs. UB 520 remnants are strikingly immunostained for either p63 (A, in pink) or CK34βE12 (B, in red), 521 independently as isolated cells or cell aggregates. In contrast, CT immunoreactivity (in green) 522 was exclusively confined to C cells of the surrounding thyroid tissue. Bar =50 µm. 523 Figure 5. Double IF for p63 and Tg (A), and CK34βE12 and Tg (B) in immature UBFs. UB 524 remnants are clearly immunostained for either p63 (A, in pink) or CK34βE12 (B, in red). Tg 525 Page 22 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vázquez Román et al. 23 immunoreactivity (in green) was mainly located at the colloid of normal thyroid follicles; 526 however, some scattered positive cells were also observed forming part of the UBF wall 527 (arrows). Furthermore, in B, a complete thyroid follicle merging, or being entrapped, from the 528 growing UBF could be observed (asterisk). Bar=25 µm. 529 Figure 6. Double immunostaining for CK34βE12 and TTF-1 in serial sections of the same 530 immature UBF. The UBF wall is clearly immunostained for CK34βE12 (cytoplasmic pattern, in 531 red), in contrast with the surrounding negative thyroid tissue. TTF-1 immunostaining (nuclear 532 pattern, in brown) was located in all differentiated thyroid cells as well as scarce cells within the 533 UBF wall that coexisted (C, arrow) or not (B, arrow) with CK positivity (B). Bar =25 µm. 534 535 Page 23 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Table 1: Antibodies used for IHC analysis. HMW CK, high-molecular weight cytokeratin; CK AE1/AE3, pan-keratin cocktail; TTF-1, thyroid transcription factor; M, monoclonal; P, polyclonal; RTU, Ready-To-Use; Ag, antigen; H, heating. Antigen Antibody Dilution Ag Retrieval HMW CK 34βE12 (M, DAKO, Denmark) RTU H CK AE1/AE3 M 3515 (M, DAKO, Denmark) 1:50 H p63 4A4 (M, Santa Cruz Biotechnology, USA) 1:500 H TTF-1 8G7G3 (M, Santa Cruz Biotechnology, USA) 1:100 H Calcitonin A0576 (P, DAKO, Denmark) 1:4000 - Thyroglobulin A0251 (P, DAKO, Denmark) 1:400 - Page 24 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Figure 6. Double immunostaining for CK34βE12 and TTF1 in serial sections of the same immature UBF. The UBF wall is clearly immunostained for CK34βE12 (cytoplasmic pattern, in red), in contrast with the surrounding negative thyroid tissue. TTF-1 immunostaining (nuclear pattern, in brown) was located in all differentiated thyroid cells as well as scarce cells within the UBF wall that coexisted (C, arrow) or not (B, arrow) with CK positivity (B). Bar =25 µm. Fig. 6 248x109mm (300 x 300 DPI) Page 31 of 31 John Wiley & Sons Journal of Morphology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60