SOME ASPECTS OF EARLY DEVELOPMENT OF THE THYMUS: EMBRYOLOGICAL BASIS FOR ECTOPIC THYMUS AND THYMOPHARYNGEAL DUCT CYST

Introduction. The aim of our morphological study is to describe the development of human thymus from 5 th up to 8 th week after fertilization in the context of its phylogenesis. We explicate some of the “forgotten” embryological terms with respect to their functions in thymic development, such as “ thymus secundus ”, “ descensus thymi ” (an embryological basis for cervical thymus) and “ ductus thymicus ” (an embryologic basis for a congenital anomaly called thymopharyngeal duct with possible thymic cyst). Material and methods. Our findings are based on the study of 18 human embryos from 6 th to 8 th week of development. Results. The first primordia of the thymus and parathyroid glands within the endoderm of pharyngeal pouches can be seen in 8 to 9 mm crown-to-rump-length stages. The most evident epithelial proliferation is visible in the paired third pharyngeal pouch ( saccus pharyngeus tertius ): the cranial dorsal part of pharyngeal pouch initiates the inferior parathyroid gland and the caudal ventral part of the pouch gives rise to the epithelial thymus. We found an obvious endodermal epithelial proliferation also in the second pharyngeal pouch. Some authors depict this proliferation as “ thymus secundus ”, but the proliferation of endoderm close down and the functional second thymus does not develop in human embryos. Conclusion. In our work we also review the clinical significance of early thymus development, as well as the most common developmental anomalies of thymus.


INTRODUCTION
In vertebrates, the pharyngeal apparatus develops from a transient series of segmental structures appearing as bulges on the cranial lateral side of the embryo, named the pharyngeal (phylogenetically so-called branchial) arches (arcus pharyngei). The pharyngeal arches are composed of tissues derived from all three embryonic germ layers. Indeed, each arch is covered by ectoderm on the outside and endoderm on its inside. Its core is of mesodermal origin and is surrounded by mesenchyme derived from neural crest (ectomesenchyme). As development proceeds, a lateral wall of the pharynx invades the arches forming an endodermal out-pocketing known as the pharyngeal pouch (saccus pharyngeus), whereas externally, the overlying ectoderm depresses forming a groove termed pharyngeal cleft (sulcus pharyngeus) (Grevellec and Tucker, 2010). The question regarding the development of organs derived from the pharyngeal region in different vertebrates belongs to one of the most complicated embryological problems. A number of studies have tried to answer the question regarding the origin of structures of the oral, pharyngeal and laryngeal cavities, original pharyngeal arches, clefts and pouches as well as organs of head and neck (Pospíšilová and Slípka, 2000;Graham, 2001;Graham, 2003;Gordon et al, 2004). The results of these studies are sometimes considerably different, because during the early ontogenesis of vertebrates not only the localization and function of pharyngeal arches, but also of ectodermal clefts and endodermal pouches change substantially (Varga et al, 2008). Phylogenetically, the pharyngeal region was the place, where the gills of aquatic vertebrates developed. The respiratory and osmoregulatory functions of gills have lost their importance in the process of evolutionary transition of animals from aquatic to terrestrial way of life. The pharyngeal (branchial) region has become adapted to the new endocrine (developing of parathyroid glands and calcitoninproducing cells of thyroid gland) and immune functions (thymus and palatine tonsil) (Slípka et al, 1998). One of the genetically determined consequences of a disrupted formation of pharyngeal apparatus is the DiGeorge´s syndrome (aplasia thymoparathyroidea) caused by a multigene-deletion from chromosome 22 (Wurdak et al, 2006). Majority of recently published manuscripts about thymus development describe its development in mice (Müller et al, 2008;Foster et al, 2008;Itoi et al, 2006), whereas classical morphological studies on human embryos and fetuses are occasional (for example Repetto et al, 2010). The aim of our morphological study is to describe the development of human thymus from 5 th up to 8 th week after fertilization in the context of its phylogenesis. We explicate some of the "forgotten" embryological terms with respect to their functions in thymic development, such as "thymus secundus", "descensus thymi" (an embryological basis for cervical thymus) and "ductus thymicus" (ductus medullaris thymi; an embryologic basis for a congenital anomaly called thymopharygeal duct with possible thymic cyst). The paired epithelial primordia of thymus derived from the third pharyngeal pouch descend together with the inferior parathyroid primordia. Influenced by "descensus thymi", the epithelial primordium detaches from the pharyngeal wall and at the beginning of the 7 th week, it may have a fissure formed in the center which can be designated as "ductus thymicus".

MATERIAL AND METHODS
Our findings are based on the study of 18 human embryos from 6 th to 8 th week of development (six embryos in each developmental week). The studied embryos belong to the collections of the Departments of Histology and Embryology of the Faculty of Medicine, Comenius University in Bratislava, Slovakia. We used serially sectioned slides, in sagittal and transversal sections. Paraffin sections (approximately seven micrometers thick) were stained with hematoxylin and eosin and with Masson´s trichrome (staining for collagen fibers). The reticular fibers were visualized by using the modified Lillie´s impregnation method (Lillie, 1965). The presence of glycogen was demonstrated by the combination of Periodoc Acid Schiff (PAS)reaction, Celestine blue and Orange G.

RESULTS
The first primordia of the thymus and parathyroid glands within the endoderm of pharyngeal pouches can be seen in 8 to 9 mm crown-torump-length stages. The most evident epithelial proliferation is visible in the third pharyngeal pouch: the cranial dorsal part of the pharyngeal pouch initiates inferior parathyroid gland and the __________________________________________________________________________________________ Todos los derechos reservados. 24 caudal ventral part of the pouch gives rise to the epithelial thymus (see Fig. 1a and 1b). The differences between these two endodermal epithelial proliferations are evident: the cells of future parathyroid gland are larger and lighter ( Fig. 2 and 3) and contain more glycogen inclusions (PAS-positive substance, Fig. 4) than the epithelial cells of developing thymus.

26
We found a quite obvious endodermal epithelial proliferation also in the second pharyngeal pouch (saccus pharyngeus secundus, Fig. 1c) in early human development (beginning of the 6 th week), but we did not find a marked epithelial proliferation in the fourth pouch (saccus pharyngeus quartus).
The paired epithelial primordia of thymus derived from the third pharyngeal pouches descend together with the third parathyroid primordia. Influenced by "descensus thymi", the epithelial primordium detaches from the pharyngeal wall and at the beginning of the 7 th week, it may have a fissure formed in the center which could be labeled as "ductus thymicus" (ductus medullaris thymi, Fig. 4).
The thymus primordia at the 7 th and 8 th week of development contain almost exclusively epithelial cells. These epithelial cells are arranged at the periphery as a row of prismatic cells, as can be seen at the light microscopic level (Fig. 5). A basal lamina separates these epithelial cells from the surrounding mesenchyme, derived from neural crest (textus cristae neuralis, mesectoderma, Fig. 6). The rapid growth of embryo causes the paired thymus primordia come close to each other and lay in front of the pericardium at the 7 th and 8 th week of development ( Fig. 7 and 8). They stay separated by a layer of connective tissue and never fuse together. In this period, the epithelial primordia of thymus ("thymus epithelialis") develop into a primary lymphoid organ ("thymus lymphaticus").

DISCUSSION
The embryonic pharynx is very important for the development of many organs, including the thyroid and parathyroid glands, palatine tonsil as well as the thymus. An epithelial proliferation of a distal part of the second, third and fourth pharyngeal pouches in the cranio-caudal sequence starts at the beginning of the sixth week of development (6 to 8 mm crown-to-rumplength). We found a quite obvious endodermal epithelial proliferation not only in the third pharyngeal pouch, but also in the second pharyngeal pouch. Some authors depict this proliferation as "thymus secundus", but the proliferation of the endoderm close down and the functional second thymus does not develop in human embryos at all (Pospíšilová and Slípka, 1994;Slípka and Pospíšilová, 1995). We assume that the thymusdeveloping potency of the second pharyngeal pouch is probably evolutionary encoded. For example, in sharks, when a thymus develops for the first time from the evolutionary point of view, the thymuses appear in all branchial clefts, except the first and the last one (Pospíšilová et al, 1999). In the process of vertebrate evolution a gradual reduction of gills together with the thymic primordia occurs. Finally, in humans, thymic primordia have been reduced usually only to the paired third pharyngeal pouch. However, according to some authors (Gasser, 1975;Van Dyke, 1941), part of thymus (thymus IV) develops from the epithelial proliferation of the fourth pharyngeal pouch. In our sections, we did not find a marked epithelial proliferation in the fourth pouch. According to Pospíšilová et al (1999), the early thymic primordia in the second and fourth pouch stop their differentiation, because they cannot fuse with the ectodermal proliferation mass of the pharyngeal clefts. This hypothesis, which supports the dual endo-and ectodermal __________________________________________________________________________________________ Todos los derechos reservados. 29 origin of epithelial thymus, was confirmed also by Rezzani et al (2008). Nevertheless, confirmed experimentally, a single pharyngeal endoderm can generate a functional thymus, too (after ectopic transplantation of pharyngeal endoderm in mice; Gordon et al, 2004). The epithelial primordium detaches from the pharyngeal wall and at the beginning of the 7 th week, it may have a "fissure" or "duct" formed in the center. This "duct" may be a reminiscence of an evolutionary stage in thymic phylogenesis in sharks Notorynchus cepedianus. In this species, all of the seven hollow thymic primordia are claimed to be joined with the branchial clefts by means of epithelial ducts. This structure confirms that at certain point the thymus had an excretory character (Slípka, 1986). The thymus primordia at the 7 th and 8 th week of development contain almost exclusively epithelial cells. The ultrastructural appearance indicates different types of epithelial cells at the periphery in comparison to those in the center of this early thymic primordium (von Gaudecker, 1986). Mass of epithelial cells that constitutes the primordial thymus attracts lymphoid precursor cells to this region from the blood-stream. The lymphoid precursor cells leave the blood vessels and migrate through the neural crest-derived mesenchyme into a primitive epithelial organ (Bockman, 1997). The disruption of thymic descent can result in an ectopic thymus (ectopia thymi) or cervical extension of thymus (textus thymicus accessorius, lobuli thymici accessorii) localized anywhere from the mandibular angle to the upper mediastinum. An ectopic thymus localized in the cervix is often called also "thymus cervicalis". The cervical thymus is a distinct entity from cervical extension of thymus. The basic difference is that in a case of ectopic thymus there is no thymus gland localized in its normal position. An ectopic thymus does not usually cause severe clinical symptoms. On the other hand, cervical remnants of thymic tissue have been found in up to 30% of infants studied at autopsy (Wagner et al, 1988). Genetic research has confirmed theory that the migration of thymic primordia is controlled by neural crest-derived cells, which are present as an ectomesenchyme on the surface of these primordia. Patients with a deletion in the HOX family of transcription factors (expressed by neural crest cells) have a normal sized thymus, but in general, it is located above its normal position (Manley and Blackburn, 2003). Various types of cervical thymus may be present as a neck mass, usually laterally, from the angle of the mandible to the manubrium. Since it is rare to diagnose this entity before its surgical removal, the differential diagnosis includes the more common pharyngeal cleft cyst, thyroglossal duct cyst, cystic hygroma, cystic dermoid and lesions of the salivary gland as well as thyroid and parathyroid glands and cervical lymph nodes (Zarbo et al, 1983). Review of literature reveals relatively few records concerning anomalies in shape and location of thymus. More than 100 cases have been described in the literature, ten percent of which occurred in neonates. Among the most severe clinical symptoms of ectopic cervical thymus belong:  Severe dyspnoe, stridor or dysphagia (Bistritzer et al, 1985;Shah et al, 2001);  Mimicking thyroid gland enlargement (Conwell and Batch, 2004) or subglottic mass as congenital haemangioma (Pai et al, 2005);  Causing snoring at sleep (Prasad et al, 2006). When ductus thymicus persists, it can be present clinically as a mass in the neck, and can develop into thymopharyngeal duct cyst -a special variant of ectopic thymic tissue (Zarbo et al, 1983). Congenital thymic cysts are uncommon and often misdiagnosed as either pharyngeal cleft cysts or cystic hygromas. However, they may have a characteristic appearance on CT. The course of the embryologic thymic tissue descent from the neck to the mediastinum indicates the potential site of deposition of an ectopic cervical thymic cyst. In a child, a cystic lesion that has an intimate relationship to the carotid sheath is likely to be a thymic cyst. Approximately 100 cases of vestigial cervical thymus or thymic cysts have been reported in children, only five of them with a persistent thymopharyngeal duct cyst (Burton et al, 1995). Other hypothesis for thymopharyngeal duct cyst development is a cystic degeneration of Hassall´s corpuscles of ectopic cervical thymus (Kaufman et al, 2001), but from an embryological point of view this hypothesis is, in our opinion, not so relevant. Thymic cysts have been described as asymptomatic and of little clinical consequence. Recent reports have stressed the possibility of respiratory compromise associated with these lesions (Wagner et al, 1988). They are usually present in the 1 st decade of life, after the age of two years, possibly because thymus reaches its greatest development before puberty. The lesions, mentioned above, may be found anywhere along the normal descent route of the thymus gland from the mandible to the manubrium sterni; 50% of them extend into the mediastinum ( Table 1. We confirm the previous findings that the most evident epithelial proliferation is visible in the third pharyngeal pouch and gives rise to inferior parathyroid glands as well as to the epithelial thymus. We found a quite obvious endodermal epithelial proliferation in the second pharyngeal pouch, too, which is probably evolutionary encoded. But we did not find a marked epithelial proliferation in the fourth pouch in our sections. According to the difficult thymic development, we summed up some of the clinical diseases related to its disruption.