SEARCH

SEARCH BY CITATION

Keywords:

  • Islet1;
  • endoderm;
  • thyroid progenitor;
  • ultimobranchial bodies;
  • C-cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The LIM homeodomain transcription factor Isl1 was investigated in mouse thyroid organogenesis. All progenitor cells of the midline thyroid diverticulum and lateral primordia (ultimobranchial bodies) expressed Isl1. This pattern persisted until the growing anlagen fused at embryonic day (E) 13.5. In Isl1 null mutants thyroid progenitors expressing Nkx2.1 and Pax8 were readily specified in the anterior endoderm but the size of the thyroid rudiment was reduced. In late development, only immature C-cells expressed Isl1. In the adult gland the number of Isl1+ cells was small compared with cells expressing calcitonin. Analysis of microarray profiles indicated a higher level of Isl1 expression in medullary thyroid carcinomas than in tumors derived from follicular cells. Together, these findings suggest that Isl1 may be a novel regulator of thyroid development before terminal differentiation of the endocrine cell types. Isl1 is an embryonic C-cell precursor marker that may be relevant also in cancer developed from the mature C-cell. Developmental Dynamics 237:3820–3829, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In higher vertebrates the thyroid is populated by two endocrine cell types that have different embryonic origins. The follicular cell progenitors that differentiate into thyroxin-producing cells in mid-late development are specified in the definitive anterior endoderm forming an outgrowth, the thyroid diverticulum or bud, which soon thereafter detaches from the pharyngeal floor and moves to the final position of the gland in the neck below the larynx. The C-cell precursors are believed to derive from the neural crest, but eventually they enter the thyroid by fusion of the midline diverticulum with the lateral thyroid primordia or ultimobranchial bodies that pinch off from the most distal pouch of the pharyngeal arches. Thus, the ultimobranchial bodies carry the presumptive C-cells to the developing thyroid before they differentiate and start to produce calcitonin. The mechanisms regulating this process and the formation of a composite gland are yet largely unknown.

Several transcription factors originally identified as regulators of thyroid-specific genes are known to play essential roles in early thyroid development. Budding and migration of the midline anlage requires the cooperation of Nkx2.1 (previously known as TTF-1), Foxe1 (previously named TTF-2), Pax8, and Hhex, which are coexpressed in the follicular progenitor cells already at the placode stage (De Felice and Di Lauro,2004). Consequently, deletion of any of these genes results in aplasia or severe dysgenesis of the gland (Kimura,1996; De Felice et al.,1998; Mansouri et al.,1998; Martinez Barbera et al.,2000). Transcription factors regulating pharyngeal arch development, e.g., Hox (Manley and Capecchi,1998), Eya1 (Xu et al.,2002), and Tbx1 (Liao et al.,2004) contribute also to the formation and maturation of the ultimobranchial bodies. Interestingly, Nkx2.1 is the only transcription factor so far known to be expressed in both the thyroid diverticulum and the ultimobranchial bodies (Mansouri et al.,1998), suggesting that mechanisms governing their development might in part follow similar regulatory pathways. Indeed, recent findings indicate that Nkx2.1 is required for the survival of both follicular and C-cell progenitors and also that Nkx2.1 haploinsufficiency leads to incomplete fusion of the primordia (Kusakabe et al.,2006).

In search for novel candidate genes implicated in thyroid organogenesis circumstantial evidence suggest that the LIM homeodomain transcription factor Islet1 (Isl1) may be of particular interest. Isl1 was originally proven to be necessary for the differentiation of exocrine and endocrine cells in the developing pancreas (Ahlgren et al.,1997); pancreatic organogenesis required Isl1 activity in both endoderm progenitors and mesenchyme surrounding the dorsal pancreatic bud. A similar expression pattern of Isl1 is evident in the anterior foregut and adjacent cardiogenic mesoderm in early mouse embryos (Cai et al.,2003). Strikingly, Isl1 deficiency causes apoptosis in the pharyngeal endoderm accompanying the severe malformations of the heart and cardiac outflow tract that leads to embryonic lethality around E10.5 (Cai et al.,2003). As thyroid progenitors assemble in this particular portion of the endoderm to form the thyroid bud, it can be hypothesized that Isl1 might also play a role in this process. This possibility is further suggested by recent findings that Isl1-regulated cardiac morphogenesis is mediated by Sonic hedgehog (Shh) expressed in the foregut endoderm (Lin et al.,2006) and that Shh homozygous mutants exhibit thyroid malformations (Fagman et al.,2004; Alt et al.,2006).

Isl1 has previously been reported to be expressed in the adult rat thyroid gland, although only C-cells showed Isl1 immunoreactivity in this study (Thor et al.,1991). By in situ hybridization, Isl1 was observed in the early thyroid placode in chicken (Yuan and Schoenwolf,2000). However, whether this is imprinted by a general Isl1 expression in the anterior endoderm or thyroid progenitors specifically express Isl1 during the developmental process is not known. We, therefore, investigated by indirect immunofluorescence the expression pattern of Isl1 in the thyroid primordia as they emerge and fuse during mouse development. In addition, we investigated whether the early thyroid bud is affected in Isl1 null embryos.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Isl1 Is Ubiquitously Expressed in the Midline Thyroid Primordium and Surrounding Embryonic Tissues

In accordance with previous reports (Lazzaro et al.,1991; Fagman et al.,2006), thyroid progenitor cells located in the endoderm lining of the pharyngeal floor were identified by immunostaining for Nkx2.1 (Fig. 1a); these cells collectively constitute the thyroid placode or midline thyroid primordium. In parallel sections, Isl1 was found to be ubiquitously expressed in the endoderm including the thyroid progenitor cells (Fig. 1b). Strong Isl1 expression was also observed in the aortic sac endothelium that closely apposed the thyroid placode and in adjacent parts of the primitive heart wall located ventrally and inferiorly of the foregut endoderm (Fig. 1b). Isl1 expression was maintained in the thyroid bud that was about to detach from the endoderm proper at E10.75 (Fig. 1c,d). At this developmental stage, the thyroid bud was entirely surrounded by Isl1-positive mesenchyme (Fig. 1d).

thumbnail image

Figure 1. A–D: Isl1 protein expression in the proximal foregut endoderm and adjacent mesoderm of mouse embryos at thyroid placode (A,B) and bud stages (C,D). A: The thyroid placode located in the pharyngeal floor close to the aortic sac is identified by Nkx2.1 expression. Epithelial cells are counterstained with E-cadherin (E-cad) antibody. B: Isl1 is ubiquitously expressed in the endoderm including the thyroid progenitor cells, and in the mesoderm of the primitive heart wall. Isl1 is weakly expressed also in the aortic sac endothelium. C,D: Nkx2.1-positive thyroid progenitor cells (C) express Isl1 (D) during evagination from the pharyngeal endoderm at embryonic day (E) 10.75. The thyroid bud is entirely surrounded by Isl1-positive mesenchyme. Arrow indicates the thyroglossal duct. A,D: Dotted line indicates contours of the aortic sac in A and of the thyroid in D. Images in A, B and C, D are, respectively, from parallel sagittal sections. Asterisk, thyroid bud; e, endoderm; tp, thyroid placode; as, aortic sac, phl, pharyngeal lumen. Scale bar = 100 μm in A–D.

Download figure to PowerPoint

Isl1 Expression Persists in the Migrating Thyroid

After degeneration of the thyroglossal duct the thyroid primordium migrates caudally along the ventral neck, reaching close association with the aortic sac at E11.5 (Fig. 2a; see also Fagman et al.,2006). Isl1 expression was maintained in the thyroid primordium as well as in the mesoderm surrounding it (Fig. 2a). This expression pattern was essentially unchanged 1 day later when the progenitor cells proliferated bilaterally to form an extended bar of parenchyma, the primitive isthmus, in front of the presumptive trachea (Fig. 2b). However, Isl1-negative mesenchyme separated the most lateral extensions of the midline thyroid from Isl1-positive mesenchyme that surrounded the trachea (Fig. 2b). Also the tracheal epithelium exhibited strong Isl1 immunoreactivity (Fig. 2b). Taken together, these observations indicate that Isl1 expression persists in the thyroid and lung primordia after completion of the budding stages.

thumbnail image

Figure 2. Isl1 expression in the detached midline thyroid primordium. A: Isl1 is expressed in the completely detached embryonic day (E) 11.5 thyroid that is located in the midline on the cranial aspect of the aortic sac. Strong Isl1 reactivity is also found in the adjacent mesenchyme. Sagittal section. B: Isl1 in the midline thyroid primordium that extends toward the ultimobranchial bodies (only visible on one side of the image due to the plane of section). The mesenchyme partly encircling the trachea as well as the airway epithelium is strongly positive for Isl1. Presence of Isl1-negative mesenchyme in unstained interstitial tissues was controlled by DAPI counterstaining of nuclei (data not shown). Transverse section. Asterisk, thyroid primordium; as, aortic sac; ub, ultimobranchial body; tr, trachea. Scale bar = 100 μm in A,B.

Download figure to PowerPoint

Thyroid Primordium Is Specified in Isl1 Null Embryos

Isl1-deficient mice are embryonic lethal at E10.5 due to severe cardiovascular developmental defects (Cai et al.,2003). In lack of a suitable conditional knockout, it was, therefore, not possible to investigate a putative role of Isl1 in the budding, migration and growth of the midline thyroid primordium. However, examination of E9.5 Isl1 null embryos revealed that Nkx2.1-positive cells appeared in the correct position in the pharyngeal endoderm (Fig. 3a,b). Their number was smaller than in corresponding wild-type littermates, and in addition the placode shape appeared slightly deteriorated. Nevertheless, immunostaining for Pax8 showed that these cells likely are the follicular progenitors (Fig. 3c). This is in agreement with previous findings on pancreas development, indicating that the specification of islet precursor cells proceeds normally in Isl1-deficient embryos (Ahlgren et al.,1997). We further found that most thyroid progenitors were closely apposed to the vessel wall of the aortic sac similar to that of the normal thyroid bud (Fig. 3a,b). This is of particular interest, because it suggests that early thyroid development might still be influenced by signal(s) generated from vessel-associated or cardiac mesenchyme, as recently shown for the embryonic thyroid in zebrafish (Fagman et al.,2004,2006; Alt et al.,2006; Wendl et al.,2007), even though Isl1 is missing.

thumbnail image

Figure 3. Specification of the thyroid placode in Isl1−/− embryos. A: Expression of Nkx2.1, marker of the thyroid placode, is detected in cells localized close to the Pecam-1–immunoreactive aortic sac in wild type embryos. B: Nkx2.1-positive cells in the anterior pharyngeal endoderm are located close to the aortic sac in Isl1 null embryos. C: Pax8 expression colocalizes with Nkx2.1 in the presumptive thyroid placode in Isl1 mutants. Parallel sections (B,C). Dotted lines indicate the apical aspect of the pharyngeal endoderm. Sagittal sections. as, aortic sac. Scale bar = 70 μm (A–C).

Download figure to PowerPoint

Isl1 Is Expressed in the Lateral Thyroid Anlagen (Ultimobranchial Bodies)

The ultimobranchial bodies, which later fuse with the midline thyroid primordium, bud from the fourth pharyngeal pouches around E10. As shown in Figure 4a, Isl1 was strongly expressed in the epithelium of these structures. The midline mesoderm investing the laryngotracheal groove also expressed Isl1, whereas lateral mesenchyme facing the ultimobranchial bodies was Isl1-negative (Fig. 4a). The ultimobranchial bodies continued to express Isl1, while the same cells gradually became Nkx2.1-positive between E10 and E11 (Fig. 4b–e). This expression pattern was essentially maintained after budding of the ultimobranchial bodies was completed at E12.5 (Fig. 5b). Notably, Nkx2.1 expression was restricted to the same endodermal structures that were positive for Isl1 (Fig. 5a). The zone of Isl1-positive mesoderm enwrapping the laryngotracheal groove had increased in size, but did not contact the ultimobranchial bodies (Fig. 5b). At E13.0, the ultimobranchial bodies were closely located to the lateral extremities of the midline primordium. Both populations of thyroid progenitor cells expressed Nkx2.1 and Isl1 (Fig. 5c,d). The same expression pattern was also found in the presumptive trachea, whereas the esophagus showed no immunoreactivity for Nkx2.1 and Isl1 (Fig. 5c,d). The trachea was almost completely surrounded by Isl1-positive mesenchyme (Fig. 5d). In addition, discrete symmetrical, wedge-shaped clusters of Isl1-expressing cells were present on both sides of the midline between the trachea and esophagus (Fig. 5d). However, the region between the midline and lateral thyroid primordia was occupied by Isl1-negative mesoderm (revealed by DAPI staining; data not shown).

thumbnail image

Figure 4. Isl1 expression in the fourth pharyngeal pouches/ultimobranchial bodies. A: Isl1 is expressed in the entire endoderm at the level of the fourth pharyngeal pouches at embryonic day (E) 10.75. Also the midline mesoderm surrounding the laryngotracheal groove (asterisk) and extending ventrally to the cardiac outflow tract is strongly positive for Isl1. B,C: Whereas only few cells express Nkx2.1 at E10.5 (B), almost all cells of the fourth pouch are positive for Isl1 already at this stage (C). D,E: At E10.75, all cells of the UB are positive for Nkx2.1 (D) and Isl1 (E; detail of A). Transverse sections. E-cadherin (green). Asterisk, midline mesoderm; oft, outflow tract. Scale bars = 100 μm in A, 30 μm in B–E.

Download figure to PowerPoint

thumbnail image

Figure 5. Isl1 in the ultimobranchial bodies after detachment from the endoderm. A: Nkx2.1 expression is restricted to the epithelium of the paired ultimobranchial bodies and the laryngotracheal groove at embryonic day (E) 12.5. B: Isl1 is expressed in the detached UB, laryngotracheal groove, and a broad zone of adjacent mesenchyme. Note that all mesoderm immediately surrounding the ultimobranchial bodies is negative for Isl1 (arrows). A,B: Parallel transversal sections. C: Before fusion at E13.0, Nkx2.1 is expressed in the trachea, ultimobranchial bodies, and midline thyroid primordium. D: Isl1 shows a similar expression pattern but is in addition strongly expressed in the mesenchyme surrounding the trachea. Note that Isl1 is not expressed in the esophagus. C,D: Parallel transverse sections. ub, ultimobranchial body; ltg, laryngotracheal groove. Scale bar = 100 μm in A–D.

Download figure to PowerPoint

Isl1 Is Down-regulated in Thyroid Follicular Progenitor Cells After Fusion of Primordia

The median and lateral thyroid anlagen fuse at E13.5. As previously detailed (Fagman et al.,2006), this process first involves growth of follicular progenitor cells over the surface of the ultimobranchial body, which thus literally is engulfed as a solid structure by the median primordium. At this stage, the ultimobranchial epithelium still showed a strong Isl1 expression (Fig. 6a). In fact, the level of immunoreactivity was outstanding in comparison to other Isl1-expressing tissues (trachea and mesenchyme). In contrast, the Isl1 expression was weak or even absent in cells derived from the midline thyroid (Fig. 6b). The loss of immunoreactivity was rapid and encompassed nearly all follicular progenitor cells, indicating that Isl1 in all probability was down-regulated rather than the cells were replaced by another cell type never expressing Isl1.

thumbnail image

Figure 6. Isl1 expression in the primitive thyroid lobes. A: After fusion at embryonic day (E) 13.5, only cells derived from the ultimobranchial bodies display strong Isl1 expression in the thyroid. Note also that the Isl1 expression level is markedly decreased in the midline mesoderm and trachea compared with the expression in the ultimobranchial body. B: Higher magnification of A showing detail of the fused primordia; arrow indicates weak or absent Isl1 immunoreactivity in progenitor cells from the midline anlage. E-cadherin (green). Transverse section. ub, ultimobranchial body; tr, trachea. Scale bars = 100 μm in A, 30 μm in B.

Download figure to PowerPoint

Isl1-Positive Cells Scatter in the Thyroid Parenchyma During Lobe Formation and Colocalize With C-cell Precursors

The primitive left and right thyroid lobes consisted of a mixture of progenitor cells derived from the two primordia that gradually merged from E13.5 and onward. However, at E15.5 remnants of the ultimobranchial bodies could still be distinguished by a weaker Nkx2.1 expression in the lobe center (Fig. 7a; see also Fagman et al.,2006). This tissue portion contained numerous Isl1-positive cells (Fig. 7b), further supporting the distinct embryonic identities of the cell populations forming the lobes. Interestingly, Isl1-positive and Isl1-negative cells were intermixed in the peripheral parts of the lobes. This pattern was more pronounced in late development (E17.5) when all Isl1-positive cells were scattered throughout the follicular parenchyma (Fig. 7d). As the distribution of Isl1-expressing cells resembled that of C-cells, we compared the localization of Isl1 with that of calcitonin. This showed that Isl1 and calcitonin immunoreactivity partly overlapped at E15.5, although the number of Isl1-expressing cells was higher than the number of calcitonin-positive cells (Fig. 7b,c). However, at E17.5, the C-cells were more numerous than the Isl1-positive cells (data not shown). Both Isl1- and calcitonin-expressing cells appeared to be integrated in the epithelial parenchyma expressing E-cadherin, although some C-cells already had attained a parafollicular position (Fig. 7e,f).

thumbnail image

Figure 7. Isl1 expression in late thyroid development. A,B: Nkx2.1 (A) and Isl1 (B) are preferentially expressed in peripheral and central regions, respectively, of the thyroid lobe at embryonic day (E) 15.5. C: Calcitonin-positive cells are mainly distributed in the same region of the lobe as Isl1 immunoreactivity. D–F: Scattered Isl1-expressing cells within the thyroid parenchyma at E17.5 (D); higher magnification of Isl1-positive cells (arrows) (E) and calcitonin (Calc) producing cells (F) in the thyroid at E17.5. E-cadherin (green). Transverse sections, parallel in A and B. pth, parathyroid. Scale bars = 100 μm in A–D, 30 μm in E,F.

Download figure to PowerPoint

Isl1-Positive Cells Are Present in the Adult Mouse Thyroid

The fact that the Isl1 expression was diminished while the C-cells started to produce calcitonin suggested that Isl1 is a C-cell precursor marker. We, therefore, investigated whether this difference prevailed in the adult thyroid. Serial sections encompassing the entire lobes were immunostained for calcitonin and Isl1, respectively. The C-cells were heterogeneously distributed throughout the gland and more often clustered around some follicles than others (Fig. 8a). Isl1-positive cells were encountered in the same tissue regions (Fig. 8b). However, the total number of cells expressing Isl1 was clearly lower than that of the C-cells. The follicular epithelium was always Isl1-negative.

thumbnail image

Figure 8. Isl1 expression in adult thyroid. A,B: Numerous calcitonin-positive cells are scattered among the follicles (A), whereas a smaller number of Isl1-positive cells are present (arrows in B). Images from parallel sections, asterisk marks the same thyroid follicle. Scale bar = 70 μm in A,B.

Download figure to PowerPoint

Isl1 Is Expressed in Medullary Thyroid Cancer

As an initial attempt to address whether Isl1 is expressed in adult human C-cells, publicly deposited microarrays were reanalyzed. Because transcriptome profiles of a pure, normal population of C-cells are not available, we focused our attention on a previously analyzed set of profiles from metastatic medullary carcinomas (MTC) in MEN2B patients (Jain et al.,2004; Ippolito et al.,2005), as these tumors derive from C-cells. As a comparison, we used a set of profiles obtained from primary papillary thyroid carcinoma of follicular subtype (PTCF; International Genomics Consortium, expO). For semiquantitative comparisons, the mean expression levels of the total genome in each data set were used as points of reference. As shown in Figure 9, transcripts for calcitonin and chromogranin A were abundant in MTC but under mean in PTCF. The reverse pattern was seen for thyroglobulin and thyroperoxidase that were highly expressed in PTCF but clearly below mean in MTC. This confirms the validity of the data sets as deriving from the expected cell types. Interestingly, whereas Isl1 transcripts were significantly above the mean expression level in MTC they were barely detectable in PTCF (Fig. 9).

thumbnail image

Figure 9. Comparison of Isl1 mRNA expression levels in one set of human medullary thyroid carcinomas (MTC, n = 9) and one set of human papillary thyroid carcinomas of follicular subtype (PTCF, n = 6). The expression level of a specific transcript is indicated as the difference from the mean expression level of all genes in each experiment. The MTC-ISL1mean is statistically different from FTC-ISL1mean (***P < 0.001). TPO, thyroperoxidase; TG, thyroglobulin; ISL1, Islet1; CHGA, chromogranin A; CALC, calcitonin.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We show in this study that Isl1 is widely expressed in the anterior foregut endoderm including the thyroid primordia that bud from, respectively, the pharyngeal floor and the fourth pharyngeal pouches in the mouse embryo. During further development, the Isl1 expression is maintained in all thyroid progenitor cells until the anlagen fuse at E13.5. Thereafter Isl1 seems to be restricted to the C-cell precursors, whereas Isl1 is down-regulated in the presumptive follicular cells. The adult thyroid contains only few Isl1-positive interstitial cells that are in clear minority to the total number of C-cells. Together, this suggests that Isl1 is a putative transcriptional regulator of both endocrine cell types in thyroid gland organogenesis.

The expression of Isl1 in the pharyngeal endoderm has previously been recognized by in situ hybridization (Cai et al.,2003). This study further showed that Isl1−/− embryos displayed decreased cell proliferation and increased apoptosis in the endoderm as well as in the subjacent splanchnic mesoderm (Cai et al.,2003), indicating that Isl1 is a survival factor to embryonic progenitor cells. Indeed, Isl1 null mice die at approximately E10.5 because of severe cardiovascular malformations related to the lack of Isl1-expressing cardiogenic progenitor cells populating the embryonic heart. It is thus feasible to assume that Isl1 expressed in early thyroid development may be important for the growth and expansion of thyroid precursors during the budding stage and thereafter. This possibility is supported by the observation that the thyroid placode had a reduced size in the Isl1 knockout as compared to Isl1+/+ E9.5 embryos. However, it should be noted that the Isl1-deficient embryo is smaller than wild-type littermates. The thyroid rudiment might thus be small for date because there is generalized growth retardation that more widely affects the endoderm and mesoderm and their organ derivatives.

As the presumptive thyroid placode cells in the Isl1-deficient embryo were immunoreactive for both Nkx2.1 and Pax8, the coexpression of which normally is confined to only follicular progenitors (De Felice and Di Lauro,2004), it is highly unlikely that Isl1 transcriptional activity is implicated in thyroid specification. This is an important conclusion, considering the fact that Isl1 was abundantly expressed also in all mesenchyme located close to the pharyngeal endoderm and the thyroid bud. Isl1+ splanchnic mesoderm in this position is likely descendant of the anterior or second heart field from which cardiac precursors building the right ventricle and the outflow tract arise (Cai et al.,2003). Recent findings suggest a role for cardiac mesoderm in early thyroid development (Wendl et al.,2007). Specifically, Nkx2.1+ thyroid progenitor cells were missing and the thyroid failed to develop in zebrafish mutants deficient of Hand2 (Wendl et al.,2007), a helix–loop–helix transcription factor implicated in heart development (Srivastava et al.,1997). As Hand2 is suggested to function downstream of Isl1 in the transcriptional network regulating the second heart field (Black,2007), it is tempting to speculate that Isl1-dependent signals generated in the pharyngeal mesoderm might influence the growth of the thyroid bud non–cell-autonomously. However, the present findings indicate that Isl1 expressed in the mesoderm (or endoderm) is not required for thyroid lineage determination in the mouse.

We found that the thyroid diverticulum continued to express Isl1 when the bud detached and migrated downward. In fact, all thyroid progenitor cells displayed strong Isl1 immunoreactivity until the fusion of primordia at E13.5. In contrast, whereas the mesenchyme surrounding the bud uniformly expressed Isl1, further growth of the midline thyroid occurred in ventral neck tissues largely devoid of Isl1. The replacement of tissue environment to Isl1-negative mesenchyme might be explained by the migration route distancing the thyroid from the cardiac mesoderm and the developing heart. However, it is also known that Isl1 is down-regulated when cardiogenic progenitor cells differentiate into distinct heart lineages (Cai et al.,2003; Moretti et al.,2007). Thus, a maintained expression of Isl1 in thyroid progenitors might designate that they are yet undifferentiated. This possibility is supported by the observation that the expression of Isl1 was lost in late development, which coincides with the onset of thyroid-specific gene expression in the mouse embryo (De Felice and Di Lauro,2004). In this respect, it is important to note that the fusion of primordia is preceded by rapid cell proliferation and tissue expansion to physically approach the anlagen and start the lobulation process (Fagman et al.,2006). Together, this suggests that the Isl1 gene is actively transcribed in several generations of the follicular progenitors before terminal differentiation.

Coexpression of Isl1 in the thyroid bud and cardiac mesoderm supports the hypothesis that thyroid and heart organogenesis may be interdependent. The likeliness of this association rests on morphological findings of a close spatiotemporal relation between the developing thyroid and the cardiac outflow tract (Hilfer and Brown,1984; Fagman et al.,2006). Moreover, there are several clinical reports on a high prevalence of cardiac malformations in children with congenital hypothyroidism caused by thyroid dysgenesis (Devos et al.,1999; Kreisner et al.,2005; Olivieri et al.,2007). Experimental evidence indicate that factors of great importance to cardiovascular development, for example, Shh (Washington Smoak et al.,2005), Tbx1 (Jerome and Papaioannou,2001; Lindsay et al.,2001; Merscher et al.,2001), and dHAND (Srivastava et al.,1997), also influence the embryonic thyroid (Fagman et al.,2004,2007; Alt et al.,2006; Wendl et al.,2007). None of these factors are expressed by the thyroid progenitor cells implicating that they act non–cell-autonomously. However, deletion of Nkx2.5, a homeobox transcription factor previously shown to be expressed in both the embryonic heart and the thyroid primordium (Lints et al.,1993), leads to developmental defects in both organs (Ikeda et al.,2002; Dentice et al.,2006). As Nkx2.5 is transcriptionally regulated by Isl1 in cardiac progenitors (Takeuchi et al.,2005), it is tempting to speculate that the same might account for Nkx2.5 in the thyroid bud. To elucidate this or other putative targets of Isl1 in thyroid development, studies on conditional mutants presently not available are required.

Isl1 was found to be ubiquitously expressed in the ultimobranchial body epithelium preceding the onset of Nkx2.1 transcription in the same cells. As these cells also are subjected to a surge of proliferation before the fusion with the median thyroid primordium takes place (Fagman et al.,2006), it is conceivable that Isl1 serves a regulatory function in the growing ultimobranchial bodies rather than being passively transmitted from the ancestral endoderm. Such a role is further supported by the notion that the ultimobranchial bodies displayed the strongest Isl1 immunoreactivity of all anterior endoderm derivatives at E12.5. In fact, as the Isl1 expression was reinforced in the ultimobranchial epithelium, it was concomitantly down-regulated in the epithelial lining of the presumptive trachea. Notably, as the ultimobranchial bodies gradually developed, they were entirely surrounded by Isl1-negative mesenchyme. This suggests that any putative Isl1-dependent mechanism in these structures follows a cell-autonomous mode of action.

During thyroid lobe formation, starting after the fusion of primordia, it was evident that cells expressing Isl1 were in clear minority scattered throughout the parenchyma. Indeed, folliculogenesis occurred solely in Isl1-negative epithelium, supporting the notion that Isl1 is down-regulated in progenitors derived from the median anlage in late development. The fact that the Isl1+ cells had the same distribution and appearance as embryonic C-cells strongly suggests that they are identical; no other thyroid cell type show this kind of tissue pattern. In higher vertebrates, C-cell precursors are carried to the embryonic thyroid by the ultimobranchial bodies by a genetically regulated mechanism involving a network of transcriptional factors that is activated in the pharyngeal pouch endoderm and associated mesenchyme (Manley and Capecchi,1995; Xu et al.,2002; Kameda et al.,2007b). Persistent expression of Isl1 in the ultimobranchial epithelium and the C-cell precursors entering the thyroid suggests a role for Isl1 in this process. The fact that Isl1 was strongly expressed in the pharyngeal pouch from which the ultimobranchial bodies bud is intriguing, considering the embryonic origin of the C-cell progenitors. According to the classic quail–chick chimera experiments by Le Dourain and co-workers more than thirty years ago (Le Douarin and Le Lievre,1970; Polak et al.,1974) the developmental source of C-cells is believed to be the neural crest that contributes with the main cellular component of the avian ultimobranchial body. However, recent genetic fate mapping studies on the progeny of Wnt1-expressing neural crest failed to demonstrate a neuroectodermal origin of mouse C-cells (Kameda et al.,2007a). Although it cannot be excluded that neural crest progenitors routed to the pharyngeal arches acquire Isl1 expression (this has been shown for neural crest cells differentiating into dorsal root and sympathetic ganglia; Ericson et al.,1992), the present data are compatible with the hypothesis that the foregut endoderm gives rise to both thyroid endocrine cell types in the mouse.

The finding that Isl1 was lost when the cells expressing calcitonin gradually increased in number between E15.5 and E17.5 is of particular importance, indicating that Isl1 is a C-cell precursor marker. This agrees well with the concept that Isl1 is a central player in embryonic programming of progenitor cells and their differentiation along specific cell lineages (Black,2007). The time preceding C-cell differentiation is characterized by multiplication of the precursors and migration into the final position dispersed among the follicles that start to form concomitantly. Conceivably, Isl1 may participate in this process before the terminal C-cell differentiation program is turned on.

Isl1+ cells were encountered in the adult thyroid, but they were scarce in comparison with the number of C-cells present in the same tissue regions. Whether this designates a subpopulation of C-cells that maintain progenitor properties in adult life is not known. However, that this may be the case is suggested from the transcription profile of medullary thyroid cancer in which the expression level of Isl1 was found to be consistently higher than in papillary cancer. Moreover, the high Isl1 level correlated with that of calcitonin and chromogranin A, which are established biochemical markers of this tumor type. The expression of Isl1 in medullary thyroid cancer might thus reflect recapitulation of some of the embryonic properties of the ancestral cell. In this respect, it is interesting to note that Isl1 recently was shown to be a novel marker of pancreatic endocrine cancer (Schmitt et al.,2008). We, therefore, propose that Isl1 might influence the proliferation, migration, and survival of malignantly transformed C-cells, analogous to the roles Isl1 exert in embryonic development (Lin et al.,2007). Future research is warranted to investigate this possibility.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Animals

C57BL/6 mice were crossed to generate wild-type embryos. The morning a vaginal plug was detected was designated 0.5 days post coitum from which estimation of embryonic age (E) was referred to. After cervical dislocation embryos were collected daily between E9.5 and E13.5 and in addition at E10.75, E15.5, and E17.5. Isl1-deficient (Isl1−/−) E9.5 embryos were kindly provided by Ulf Ahlgren, UCMM, Umeå University. Genotyping was performed as described in (Pfaff et al.,1996). Adult thyroid glands still connected to the larynx and trachea were dissected immediately after the animals were killed with carbon dioxide. Animal handling and experiments were approved by the local ethic committee at the University of Gothenburg.

Immunoreagents

The following antibodies were used for immunofluorescent staining: Rat mAb against E-cadherin (ECCD-2, Calbiochem, La Jolla, CA), rat mAb against PECAM-1 (Pharmingen, Stockholm, Sweden), rabbit pAb against Nkx2.1 (Biopat, Milan, Italy), rabbit pAb against calcitonin (DAKO, Glostrup, Denmark), rabbit pAb against Islet1 (kindly provided by prof. Helena Edlund, UCMM, Umeå, Sweden), rabbit pAb against Pax-8 (Postiglione et al.,2002; kindly provided by Prof. Roberto di Lauro), and biotin-conjugated anti-rat and rhodamine red-X-conjugated anti-rabbit IgGs (Jackson ImmunoResearch, West Grove, PA). Streptavidin–fluorescein isothiocyanate (FITC) was purchased from DAKO. The specificity of the Isl1 antibody has previously been demonstrated (Thor et al.,1991).

Immunohistochemistry

Embryos and adult thyroids were immersion-fixed in 4% paraformaldehyde overnight at 4°C. To allow sufficient fixation of E12.5 and older embryonic specimens, the embryos were decapitated at the level of the nose (i.e., cranial to the neck region of interest) and removed from the inferior half of the trunk. For cryoprotection, the tissue samples were incubated overnight at 4°C in 30% sucrose before being embedded in Tissue Tek (Sakura, Zoeterwoude, the Netherlands) and frozen at −80°C.

Ten-micrometer-thick sections were cut on a cryostat microtome (Microm HM 500M) and collected on polylysine glass slides (Menzel-Gläser, Braunschweig, Germany). To improve the adherence of sections from E17.5 embryos super-frosted glass slides (Menzel-Gläser) were used. Air-dried sections were permeabilized by incubation with 0.1% Triton X-100 for 20 min, blocked in phosphate buffered saline with 2% normal donkey serum (Jackson ImmunoResearch) for 1 hr at room temperature, and then incubated overnight at 4°C with primary antibodies diluted in blocking buffer. Immunolabeled sections were incubated with secondary antibodies also diluted in blocking buffer for 1 hr and thereafter with Streptavidin-FITC for 30 min at room temperature. All incubation steps were followed by washing in 0.1% Triton X-100 for 3 × 5 min. Microscopy and imaging were done in a Nikon Microphot FXA epifluorescence microscope equipped with a QLC100 confocal laser scanning module (VisiTech International, Sunderland, UK) or a Bio Radiance 2000 Laser Scanning Microscope. Images were processed using the Image Pro Plus software (Media Cybernetics, Silver Spring, MD), ImageJ, and Adobe Photoshop Elements 3.0.

Microarray Data Analysis

Raw data from two publicly deposited gene expression profiling experiments available in .cel format were reanalyzed. The first data set (Jain et al.,2004; Ippolito et al.,2005) included samples from 11 medullary thyroid carcinomas (MTC, primary and metastatic) from MEN2B patients, which have been hybridized on Affymetrix GeneChip HG-U95Av2 arrays. The second data set (International Genomics Consortium, expO) included samples from 9 papillary thyroid carcinomas (follicular variant; PTCF), which have been hybridized on Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Because the expression profile data were derived from two different experiments using different platforms they could not be directly compared in a single experiment. Gene expression values were, therefore, compared using the GeneSpringGX 9.0.5 RMA summarization algorithm without per gene normalization, because two different parameters were not compared, for each experiment and then expressed in log2 mode. Preliminary inspection of individual expression profiles of thyroglobulin and thyroperoxidase was then performed. Three PTCF samples with low expression levels of thyroglobulin and thyroperoxidase, suggesting a lower degree of differentiation, were, therefore, excluded from further analysis. In the MTC group two samples differed significantly from the remaining nine in that they expressed high levels of thyroglobulin and thyroperoxidase. Because it was suspected that these two samples could represent mixed medullary-follicular carcinomas (Kostoglou-Athanassiou et al.,2004) or a contribution of normal thyroid tissue in the sample, they were excluded from further analysis. For comparisons of expression levels of individual transcripts between groups, the mean expression of all genes in each data set was subtracted from the expression value of the transcript of interest. Expression values are thus reported as differences from mean. Statistical analysis of data was performed using the t-test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Prof. Helena Edlund for generously sharing the Isl1 antibody and members of the Ulf Ahlgren laboratory for providing Isl1 knockout embryos. We also thank the Centre for Cellular Imaging (CCI) at Sahlgrenska Academy, University of Gothenburg, for technical assistance and use of the Bio Radiance 2000 Laser Scanning Microscope. H.F. was supported by an EMBO long-term fellowship.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Ahlgren U, Pfaff SL, Jessell TM, Edlund T, Edlund H. 1997. Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells. Nature 385: 257260.
  • Alt B, Elsalini OA, Schrumpf P, Haufs N, Lawson ND, Schwabe GC, Mundlos S, Gruters A, Krude H, Rohr KB. 2006. Arteries define the position of the thyroid gland during its developmental relocalisation. Development 133: 37973804.
  • Black BL. 2007. Transcriptional pathways in second heart field development. Semin Cell Dev Biol 18: 6776.
  • Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J, Evans S. 2003. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell 5: 877889.
  • De Felice M, Ovitt C, Biffali E, Rodriguez-Mallon A, Arra C, Anastassiadis K, Macchia PE, Mattei M-G, Mariano A, Scholer H, Macchia V, Di Lauro R. 1998. A mouse model for hereditary thyroid dysgenesis and cleft palate. Nat Genet 19: 395398.
  • De Felice M, Di Lauro R. 2004. Thyroid development and its disorders: genetics and molecular mechanisms. Endocr Rev 25: 722746.
  • Dentice M, Cordeddu V, Rosica A, Ferrara AM, Santarpia L, Salvatore D, Chiovato L, Perri A, Moschini L, Fazzini C, Olivieri A, Costa P, Stoppioni V, Baserga M, De Felice M, Sorcini M, Fenzi G, Di Lauro R, Tartaglia M, Macchia PE. 2006. Missense mutation in the transcription factor NKX2–5: a novel molecular event in the pathogenesis of thyroid dysgenesis. J Clin Endocrinol Metab 91: 14281433.
  • Devos H, Rodd C, Gagne N, Laframboise R, Van Vliet G. 1999. A search for the possible molecular mechanisms of thyroid dysgenesis: sex ratios and associated malformations. J Clin Endocrinol Metab 84: 25022506.
  • Ericson J, Thor S, Edlund T, Jessell TM, Yamada T. 1992. Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256: 15551560.
  • Fagman H, Grande M, Gritli-Linde A, Nilsson M. 2004. Genetic deletion of Sonic Hedgehog causes hemiagenesis and ectopic development of the thyroid in mouse. Am J Pathol 164: 18651872.
  • Fagman H, Andersson L, Nilsson M. 2006. The developing mouse thyroid: embryonic vessel contacts and parenchymal growth pattern during specification, budding, migration, and lobulation. Dev Dyn 235: 444455.
  • Fagman H, Liao J, Westerlund J, Andersson L, Morrow BE, Nilsson M. 2007. The 22q11 deletion syndrome candidate gene Tbx1 determines thyroid size and positioning. Hum Mol Genet 16: 276285.
  • Hilfer SR, Brown JW. 1984. The development of pharyngeal endocrine organs in mouse and chick embryos. Scan Electron Microsc (pt 4): 20092022.
  • Ikeda Y, Hiroi Y, Hosoda T, Utsunomiya T, Matsuo S, Ito T, Inoue J, Sumiyoshi T, Takano H, Nagai R, Komuro I. 2002. Novel point mutation in the cardiac transcription factor CSX/NKX2.5 associated with congenital heart disease. Circ J 66: 561563.
  • Ippolito JE, Xu J, Jain S, Moulder K, Mennerick S, Crowley JR, Townsend RR, Gordon JI. 2005. An integrated functional genomics and metabolomics approach for defining poor prognosis in human neuroendocrine cancers. Proc Natl Acad Sci U S A 102: 99019906.
  • Jain S, Watson MA, DeBenedetti MK, Hiraki Y, Moley JF, Milbrandt J. 2004. Expression profiles provide insights into early malignant potential and skeletal abnormalities in multiple endocrine neoplasia type 2B syndrome tumors. Cancer Res 64: 39073913.
  • Jerome LA, Papaioannou VE. 2001. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat Genet 27: 286291.
  • Kameda Y, Nishimaki T, Chisaka O, Iseki S, Sucov HM. 2007a. Expression of the epithelial marker E-cadherin by thyroid C cells and their precursors during murine development. J Histochem Cytochem 55: 10751088.
  • Kameda Y, Nishimaki T, Miura M, Jiang SX, Guillemot F. 2007b. Mash1 regulates the development of C cells in mouse thyroid glands. Dev Dyn 236: 262270.
  • Kimura S. 1996. Thyroid-specific enhancer-binding protein: role in thyroid function and organogenesis. Trends Endocrinol Metab 7: 247252.
  • Kostoglou-Athanassiou I, Athanassiou P, Vecchini G, Gogou L, Kaldrymides P. 2004. Mixed medullary-follicular thyroid carcinoma. Report of a case and review of the literature. Horm Res 61: 300304.
  • Kreisner E, Neto EC, Gross JL. 2005. High prevalence of extrathyroid malformations in a cohort of Brazilian patients with permanent primary congenital hypothyroidism. Thyroid 15: 165169.
  • Kusakabe T, Hoshi N, Kimura S. 2006. Origin of the ultimobranchial body cyst: T/ebp/Nkx2.1 expression is required for development and fusion of the ultimobranchial body to the thyroid. Dev Dyn 235: 13001309.
  • Lazzaro D, Price M, de Felice M, Di Lauro R. 1991. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 113: 10931104.
  • Le Douarin N, Le Lievre C. 1970. [Demonstration of neural origin of calcitonin cells of ultimobranchial body of chick embryo]. C R Acad Sci Hebd Seances Acad Sci D 270: 28572860.
  • Liao J, Kochilas L, Nowotschin S, Arnold JS, Aggarwal VS, Epstein JA, Brown MC, Adams J, Morrow BE. 2004. Full spectrum of malformations in velo-cardio-facial syndrome/DiGeorge syndrome mouse models by altering Tbx1 dosage. Hum Mol Genet 13: 15771585.
  • Lin L, Bu L, Cai CL, Zhang X, Evans S. 2006. Isl1 is upstream of sonic hedgehog in a pathway required for cardiac morphogenesis. Dev Biol 295: 756763.
  • Lin L, Cui L, Zhou W, Dufort D, Zhang X, Cai C-L, Bu L, Yang L, Martin J, Kemler R, Rosenfeld MG, Chen J, Evans SM. 2007. 2-Catenin directly regulates Islet1 expression in cardiovascular progenitors and is required for multiple aspects of cardiogenesis. Proc Natl Acad Sci U S A 104: 93139318.
  • Lindsay EA, Vitelli F, Su H, Morishima M, Huynh T, Pramparo T, Jurecic V, Ogunrinu G, Sutherland HF, Scambler PJ, Bradley A, Baldini A. 2001. Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410: 97101.
  • Lints TJ, Parsons LM, Hartley L, Lyons I, Harvey RP. 1993. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119: 419431.
  • Manley NR, Capecchi MR. 1995. The role of Hoxa-3 in mouse thymus and thyroid development. Development 121: 19892003.
  • Manley NR, Capecchi MR. 1998. Hox group 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands. Dev Biol 195: 115.
  • Mansouri A, Chowdhury K, Gruss P. 1998. Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet 19: 8790.
  • Martinez Barbera JP, Clements M, Thomas P, Rodriguez T, Meloy D, Kioussis D, Beddington RS. 2000. The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation. Development 127: 24332445.
  • Merscher S, Funke B, Epstein JA, Heyer J, Puech A, Lu MM, Xavier RJ, Demay MB, Russell RG, Factor S, Tokooya K, Jore BS, Lopez M, Pandita RK, Lia M, Carrion D, Xu H, Schorle H, Kobler JB, Scambler P, Wynshaw-Boris A, Skoultchi AI, Morrow BE, Kucherlapati R. 2001. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104: 619629.
  • Moretti A, Lam J, Evans SM, Laugwitz KL. 2007. Biology of Isl1+ cardiac progenitor cells in development and disease. Cell Mol Life Sci 64: 674682.
  • Olivieri A, Medda E, De Angelis S, Valensise H, De Felice M, Fazzini C, Cascino I, Cordeddu V, Sorcini M, Stazi MA, The Study Group for Congenital H. 2007. High risk of congenital hypothyroidism in multiple pregnancies. J Clin Endocrinol Metab 92: 31413147.
  • Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM. 1996. Requirement for LIM homeobox gene Isl1 in Motor neuron generation reveals a motor neuron-dependent step in interneuron. Differentiation Cell 84: 309320.
  • Polak JM, Pearse AG, Le Lievre C, Fontaine J, Le Douarin NM. 1974. Immunocytochemical confirmation of the neural crest origin of avian calcitonin-producing cells. Histochemistry 40: 209214.
  • Postiglione MP, Parlato R, Rodriguez-Mallon A, Rosica A, Mithbaokar P, Maresca M, Marians RC, Davies TF, Zannini MS, De Felice M, Di Lauro R. 2002. Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland. Proc Natl Acad Sci U S A 99: 1546215467.
  • Schmitt AM, Riniker F, Anlauf M, Schmid S, Soltermann A, Moch H, Heitz PU, Kloppel G, Komminoth P, Perren A. 2008. Islet 1 (Isl1) expression is a reliable marker for pancreatic endocrine tumors and their metastases. Am J Surg Pathol 32: 420425.
  • Srivastava D, Thomas T, Lin Q, Kirby ML, Brown D, Olson EN. 1997. Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nat Genet 16: 154160.
  • Takeuchi JK, Mileikovskaia M, Koshiba-Takeuchi K, Heidt AB, Mori AD, Arruda EP, Gertsenstein M, Georges R, Davidson L, Mo R, Hui C-c, Henkelman RM, Nemer M, Black BL, Nagy A, Bruneau BG. 2005. Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development. Development 132: 24632474.
  • Thor S, Ericson J, Brannstrom T, Edlund T. 1991. The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat. Neuron 7: 881889.
  • Washington Smoak I, Byrd NA, Abu-Issa R, Goddeeris MM, Anderson R, Morris J, Yamamura K, Klingensmith J, Meyers EN. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev Biol 283: 357372.
  • Wendl T, Adzic D, Schoenebeck JJ, Scholpp S, Brand M, Yelon D, Rohr KB. 2007. Early developmental specification of the thyroid gland depends on han-expressing surrounding tissue and on FGF signals. Development 134: 28712879.
  • Xu P-X, Zheng W, Laclef C, Maire P, Maas RL, Peters H, Xu X. 2002. Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid. Development 129: 30333044.
  • Yuan S, Schoenwolf GC. 2000. Islet-1 marks the early heart rudiments and is asymmetrically expressed during early rotation of the foregut in the chick embryo. Anat Rec 260: 204207.