Developmental events and cellular changes occurred during esophageal development of quail embryos

The current study focused on the histogenesis of the esophagus in quail embryos. Formation of the gut tube occurred on the 4th day of incubation. Development of the muscular layers occurred in a sequential manner; the inner circular layer on the 7th day, the outer longitudinal layer on the 8th day and the muscularis mucosae on the 9th day. Glandular development began on the 13th day of incubation. The epithelium was pseudostratified columnar that consisted of mucous cells, dendritic cells, and keratinocyte precursors. Epithelial stratification occurred on the 15th day of incubation. We used Mallory trichrome, Weigert-Van Gieson, and Gomori silver stains to visualize fibrous components. Scanned samples showed formation of endoderm and mesoderm on the 5th day of incubation. A layer of myoblasts developed on the 8th day of incubation. Formation of mucosal folds, which contained glandular openings, occurred on the 14th to 17th days of incubation. On the 5th to 8th days of incubation, CD34 and vascular endothelial growth factor (VEGF) positive-mesodermal cells, and telocytes (TCs) were detected. On the 15th day of incubation, CD34 and VEGF positive-telocytes, and fibroblasts, were identified. The current study described the correlations between functional morphology and evolutionary biology.


Table1.
The processing time of the samples in paraffin embedding techniques. NBF (neutral buffer formalin), h hours, d days, MB I methyl bonzoate1, MB II methyl benzoate II, PI paraffin I, P II paraffin II, P III paraffin III.

1-F A-NBF 24 h
B_Bouin's solution 1/2 h www.nature.com/scientificreports/ HRP/DAB manufactured by Thermo Fisher Scientific TP-015HD). The procedures were performed according to the manufacturer's instructions [19][20][21] . Paraffin sections measuring 5 µm were dewaxed by xylene, hydrated with ascending grades of alcohol, and washed for 5 min twice with PBS (phosphate buffered saline) at pH 7.4 ( Table 1). The sections were treated with 3% hydrogen peroxide in methanol at room temperature for 20 min to block endogenous peroxidase activity. The sections were rinsed for 10 min with running tap water. Improving antigen retrieval requires use of a 10 mm sodium citrate buffer (pH 6.0) ( Table 2) in a water bath for 20 min at 95-98 °C. This was followed by slide cooling for 20 min at room temperature, and subsequently washed for 5 min twice with PBS at pH 7.4. Ultra V block was used for 5 min at room temperature to prevent nonspecific background staining. To avoid artifacts, use of ultra V block should not exceed 10 min. The primary antibody (Table 3) was applied on the sections overnight at 4 °C. The sections were then washed for 5 min thrice with PBS at pH 7.4. The biotinylated secondary antibody (Goat Anti-Polyvalent, Anti-Mouse IgG + Anti -Rabbit IgG; Thermo Fisher Scientific, UK; Lab Vision Corporation; Table 2) was applied on the sections for 10 min at room temperature. The sections were then washed for 5 min twice with PBS at pH 7.4 and incubated for 10 min at room temperature using streptavidin-peroxidase complex (Thermo Fisher Scientific, UK; Lab Vision Corporation, USA). Visualization of the bound antibodies was carried out by incubating the section in a humid chamber using a mixture of 1 drop of 3,3′-diaminobenzidine (DAB) and chromogen (  Table 3. Identity, sources, and working dilution of antibodies used in immunohistochemical studies. Antibodies used that showed reactivity in Avian species. Immunohistochemical procedures of vascular endothelial growth factor (VEGF). Samples used for VEGF immunostaining were fixed in NBF (neutral buffer formalin) ( Table 1). The two-step immunohistochemical staining procedures utilized the DAKO EN Vision TM and System, HRP peroxidase 22 . The staining was performed according to instructions 23 . Paraffin sections of 5 µm were dewaxed, rehydrated, and rinsed for 5 min twice with PBS at pH 7.4. Blocking of the endogenous peroxidase activity was achieved by using drops of 3% hydrogen peroxide in methanol for 20 min at room temperature, then thoroughly washed with running tap water for 10 min. Antigen retrieval was performed using 10 mm sodium citrate buffer (pH 6.0) ( Table 2). The buffer was heated to 95 °C-98 °C in a water bath for 20 min, followed by cooling at room temperature for 20 min. Sections were washed for 5 min twice with PBS at pH 7.4. Blocking nonspecific background staining was performed using drops of blocking serum (DAKO) to cover the sections for 5 min at room temperature. The sections were then incubated with the primary antibody. The antibodies that were applied showed immunoreactivity in avian species 24 . Table 3 explored the identity, sources, and the working dilution of antibodies used in the immunohistochemical technique. The slides were rinsed for 5 min thrice with PBS at pH 7.4 then incubated with secondary antibody for 30 min at room temperature. The slides were again washed for 5 min twice with PBS at pH 7.4 and incubated for 5-10 min at room temperature with 3,3′-diaminobenzidine (DAB) and substrate-chromogen that produced a brown color at the antigen site. The slides were then counterstained with Harris hematoxylin for 30 s. The sections were dehydrated with ethanol alcohol 90%, then 100% II, cleared in xylene, and covered using DPX. VEGF immunohistochemically-stained sections were examined using the Leitz Dialux20 microscope provided with the Canon (PowerShot A95) digital camera -vecontrols were performed with the same procedures except using primary antibodies.

Preparation of samples for embedding in resin.
Resin embedding technique was performed according to 25 using Karnovsky's fixed samples. Five samples were used from incubation days 5, 8, and 15. Each esophagus, measuring 2-3 mm in length, was carefully excised after sacrificing the quail. Karnovsky fixative 26 was prepared from a mixture of 10 mL of 25% paraformaldehyde, 10 mL of 50% glutaraldehyde, 50 mL phosphate buffer, and 30 mL distilled water. Karnovsky fixative was applied overnight at 4 °C. The samples were postfixated by osmium tetroxide, dehydrated using ascending grades of alcohol, inoculated in a mixture of alcohol/ resin and pure resin, resin embedding and crystallization was performed in oven at 60 °C degrees. Semi-thin sections (1 μm) were created using an ultramicrotome (Ultracut E, Reichert-Leica, Germany) and stained with toluidine blue 27,28 , methylene blue 29,30 , and periodic acid-Schiff (PAS) 31 . Staining of semi-thin sections required dissolving of resin using saturated alcoholic solution of sodium hydroxide. The stained sections were examined using a Leitz Dialux 20 microscope and a Canon digital camera (Canon PowerShot A95).

Results
On the 4th day of incubation, the gut tube was formed and consisted of endoderm and covered by mesoderm (Fig. 1A,B). On the 5th day of incubation, the primitive esophagus had an endodermal layer of pseudostratified epithelium and surrounded by a condensed layer of mesenchyme, a marker for the development of a muscular wall (Fig. 1C,D). This muscular wall began to develop on the 6th day of incubation. A circular layer of SMC (smooth muscle cells) was observed (Fig. 1E,F). On the 7th day of incubation, the circular muscular layer became more distinct while myoblasts aggregated to form the primitive outer longitudinal muscular layer ( Fig. 2A-C).
On the 8th and 9th days of incubation, the esophagus developed distinct inner circular and outer longitudinal muscular layers (Figs. 2A-I). The muscularis mucosae recognized on the 8th day of incubation by mesenchymal condensation and identified as a thin layer of SMC that supports the mucosa on the 9th day of incubation. (Fig. 2H). On the 13th day of incubation, the esophagus developed muscualris mucosae rather than the distinctive inner circular and outer longitudinal muscular layers ( Fig. 3A-D). The epithelial invaginations formed sac-like glandular units. The epithelium was pseudostratified type (Fig. 3A,B). Presence of collagen fibers in the lamina propria, between the muscle fibers, and the serosa were visualized using Mallory trichrome (Fig. 3C) and Weigert-Van Gieson stains (Fig. 3D,E). On the 15th day of incubation, reticular fibers were recognized using Gomori's silver stain in the lamina propria (Fig. 4A,B), around the gland (Fig. 4D), around smooth muscle cells and in the myenteric or Auerbach's plexus (Fig. 4E,F). TCs were identified in the LP (Fig. 4C) and between the muscle bundles ( Fig. 4G-I). www.nature.com/scientificreports/ Bielschowsky's silver stain was used to visualize the SMC, which revealed a granular appearance indicating the presence of dense bodies ( Fig. 4G-I). The thoracic portion of the esophagus developed highly folded mucosa which exhibited numerous sac-like glandular units, inactive glands ( Fig. 5A,B) the epithelial lining had stratified squamous epithelium non-keratinized (Fig. 5C). The cervical portion also had stratified squamous epithelium non-keratinized developed active mucous esophageal glands ( Fig. 5D-F). Telocytes were recognized in the lamina propria ( Fig. 5E) and submucosa (Fig. 5F). The pseudostratified epithelium and the basal lamina appreaed PAS-positive on the 8th day of incubation ( Fig. 6A-C). The developing muscular layers were distinguished by the myoblasts (Fig. 6D), some of which contained PAS-positive glycogen granules. PAS-positive myelin sheaths were also detected between myoblasts (Fig. 6D). On the 15th day of incubation, the thoracic portion of the esophagus was lined by pseudostratified epithelium exhibited PAS activity while the gland was inactive PAS-ve (  www.nature.com/scientificreports/ contained interstitial cells rich in dilated RER (Fig. 8G). The developing muscular layers were distinguished by the myoblasts (Fig. 8H). On the 17th day of incubation, the esophageal keratinocytes developed keratin intermediate filaments (Fig. 9A,B). Dendritic cells appeared between keratinocytes. Dendritic cells were recognized by vesicles, www.nature.com/scientificreports/ multivesicular bodies, dense granules, and rod-shaped granules (Fig. 9C). Esophageal glands contained mucous granules, RER, and exhibited interdigitation (Fig. 9D,E). Collagen fibers were identified using TEM (Fig. 9A,B,F). On the 5th day of incubation, scanned samples of the esophagus showed the presence of endoderm and mesoderm and the endoderm was pseudostratified epithelium (Fig. 10A,B). On the 8th day of incubation, the pseudostratified epithelium was also existed (Fig. 10C-F). Development of the myoblast layer occurred at this age ( Fig. 10E-H). On the 14th day of incubation, the esophagus formed a distinct mucosal fold which contained the opening of the esophageal glands ( Fig. 10I-L), and on the 17th day of incubation (Fig. 10Q,S,T) the esophageal glands were now visible in the lamina propria (Fig. 10R). TCs were identified in the muscular layer ( Fig. 10 M-P).
On the 5th day of incubation, CD34 and VEGF positive cells were identified in the subepithelial tissue and the mesenchyme. CD34 positive TCs were distinguished by cell prolongations or telopodes and VEGF positive TCs were recognized as well (Fig. 11A,B). On the 8th day of incubation, it is notable that CD34 positive cells were more obvious in the lamina propria compared to VEGF. CD34 and VEGF positive cells were also identified in the peri-muscular tissue. CD 34 positive and VEGF positive TCs were distinguished by their telopodes (Fig. 12A-C). The sprouting endothelial cells were CD34 positive (Fig. 13E). Figure 13 F represented negative control for CD34.
On the 15th day of incubation, Interstitial cells in the lamina propria and the submucosa were CD34 positive including TCs that had distinct telopodes, and fibroblasts which had voluminous cytoplasm and short cell processes (Fig. 13A-D). Perivascular tissue between muscles had strong CD34 immunoaffinity (Fig. 13D). CD34positive TCs were detected between muscle fibers and bundles (Fig. 13D). Interstitial fibroblast-like cells in the lamina propria were VEGF positive (Fig. 14A). Endothelial sprouting in the lamina propria were VEGF positive (Fig. 14B). VEGF-positive TCs were identified in the lamina propria and the submucosa (Fig. 14C,D). Perivascular tissue had strong immunoaffinity for VEGF (Fig. 14E). Figure 14 F represented negative control for VEGF.
All events that occurred during esophageal development were summarized in (Fig. 15). www.nature.com/scientificreports/

Discussion
The current study investigated histological events occurring during the development of the esophagus of the quail. The gut tube was formed on the 4th day of incubation. This was composed of endoderm and covered by mesoderm, which was previously described by another study 52 . The authors mentioned the vacuolated stage, in which extracellular vacuoles appeared between the epithelial cells, and they suggested that the vacuoles may be related to the developmental cysts.
Mesenchymal condensation was the first sign of development of the muscular layer. Development of the muscular layer occurred in a sequential manner; the inner circular layer on the 7th day, the outer longitudinal layer on the 8th day, and the muscularis mucosae began by mesenchymal condensation and development of strands of SMC on the 9th day. A similar sequence of muscular development occurs in the pheasant esophagus; development of the inner circular layer of the begins on the 8th day, the outer longitudinal layer on the 12th day, and the muscularis mucosae is completed on the 18th day 53 . The sequence of muscular coat development of the human embryos is also similar to avian species. However, in the Guinea Fowl (Numida meleagris). The tunics of the muscularis are not well developed till at posthatch 54 . The muscular layers of the human esophagus develop in similar sequence to quail. In the 12 mm and 20 mm crown-rump stages human embryos, the inner muscular layer become more developed. The outer longitudinal muscle layer and the muscularis mucosae are prominent 55 .
In the current study, PAS-positive inclusions were detected in some myoblasts and myelin sheaths. PAS is used to identify the simple sugars in myelin 56 . The study used Bielschowsky's silver stain to visualize the SMC, which exhibited a granular appearance indicating dense bodies. Visualization of the myofilaments striations in skeletal muscles was documented by using Bielschowsky's stain 57 .
Esophageal epithelium directly transformed from pseudostratified columnar into the stratified squamous non-keratinized type by the 15th day. In all stages of esophageal development, the epithelium was non-ciliated that may be a unique feature of quail esophagus. A different types of epithelium is noted in the pheasant; pseudostratified columnar epithelium is observed on the 9th day of incubation and transformed into stratified cuboidal by the 10th day, then to simple cuboidal epithelium between the 10th and 12th days, and finally to stratified squamous on the 13th day 53 . In the chukar partridge, the epithelium transformed to 2 layered cuboidal cells with some cilia by day 9 of incubation. The ciliated cells increased during embryonic development on day 10, 11. By day 18 of incubation, two types of simple and pseudostratified columnar epithelial tissue are detectable in the thoracic portion of esophagus. The cilia were difficult to distinguished due to the epithelial secretion 58   www.nature.com/scientificreports/ ends. The ciliated cells gradually disappear with proceeding the development and only isolated patches of ciliated columnar cells occasionally remain until after birth 55 . Development of ciliated esophageal epithelium is associated with transformation into stratified columnar in type. Formation of ciliated stratified columnar epithelium occurs on the 8th week of gestation and were decreased after the14th week. The stratified columnar epithelium transforms into the stratified squamous epithelium during the 4th month 59 . Glandular development began on the 13th day of incubation. Epithelium invaginated into the underlying connective tissue, forming sac-like glandular units. A similar occurrence can be observed in chickens, starting on the 12th day of incubation 60 . In pheasants, glandular development begins as epithelial buds in the esophagus on the 18th day of incubation 53 . The current study revealed epithelial transformation preceded glandular activation. The undifferentiated pseudostratified epithelial cells the differentiated into keratinocytes and dendritic cells. Esophageal glands which synthesize polysaccharide components were detected using PAS and toluidine blue. Through the metachromatic reaction from toluidine blue, some of these components were identified as glycosaminoglycans (GAGs), which the avian esophageal glands secrete along with mucopolysaccharides 53,60 . The submucosa of the quail esophagus was a delicate layer. Similar to other avian species, very thin layer of submucosa is described in the pariah kite, median egret, goshawk, dove and duck 61 , the House Sparrow 62 , and chukar partridge (Alectoris chukar) 58 . In pigeons, the submucosa is thin in female (40.2±7.5 in the cervical region and 20.6± 3.6 in the thoracic region) and is relatively thicker in male (60.7±11.5 in the cervical region and 40.6±8.9 in the thoracic region) 63 . While, the submucosa in the esophagus of human 64 and small animals 65 is a thick layer.
In the current study, we focused on the morphological novelties of quail esophagus and discussed the possible role in adaptation to their environments. Embryonic esophageal epithelium of quail didn't acquire cilia. Unlike, other avian and mammalian species 55,58,59 . The development of the cilia is related to the functional requirements. It is not clear whether the cilia are motile true cilia that remove the surface secretions/foreign www.nature.com/scientificreports/ particles or non-motile stereocilia that has absorptive function. In chick embryos, the esophageal cilia associate with microvilli and microplicae 66 . Esophageal cilia have been described as transitory structures in avian and mammals in contrast to the lower vertebrates such as the crocodiles. Esophagus of the juvenile American alligators (Alligator mississippiensis) has ciliated columnar epithelium 67 . It is interesting that embryonic quail esophagus lacked the skeletal muscles, unlike the higher (mammalian) and lower species (aquatic and reptiles). The esophagus is devoid of striated muscles in wild avian species such as Rock Dove, Collared Dove, Rose-ringed Parakeet, Kestrel, House Sparrow and Linnet 68 , mallard, spot-billed duck, Ural owl and Hodgson's hawk-eagle 69 , and other birds, Kingfisher (Halcyon smyrnensis) and Hoopoe (Upupa epops) 70 and chicken 71 . Thus, it seems that involuntary control of the esophagus is unique feature of avian species. This may be correlated to feeding habitat that requires peristatic involuntary movement during regurgitative feeding to feed their young. It seems that regurgitation may require definite time lapsing to deliver a semi-digested food. In mammals, the muscular wall of the esophagus is comprised of both skeletal and smooth muscles. However the distribution of the two types varies according to animal species 72,73 . Among fish, the esophagus of Anablepsoidesurophthalmus has some striated muscle fibers 74 . While, heavy striated musculature thick inner longitudinal and outer circular layer are described in tilapia 75 , Trachelyopterus striatulus (Siluriformes: Auchenipteridae) 76   www.nature.com/scientificreports/ of the esophageal muscular layer with the variable degree of esophageal striated myogenesis. Esophageal striated muscle progenitor cells that originate in the craniopharyngeal mesoderm and express the early marker Mesp1, and subsequently express Tbx1 and Isl1. Esophageal striated muscle progenitor cells colonize the proximal portion of the esophageal muscular layer. They migrate distally along the esophageal muscular layer forming the transition zone where they express Pax7, Myf5 and MyoD, and myogenin. Some Pax7 + and Myf5 + /MyoD + cells propagate to generate adequate numbers of precursor cells for the muscular layer. Some differentiate into striated myofibers, which form proximal to the transtion zone. The authors descried a linear expression pattering Isl1, Pax7, Myf5/MyoD progression 77 . The architecture of the esophageal glands of avian and mammals are quite similar. Mammalian esophageal glands are compound tubuloalveolar type in mammals 73 and some birds such as barn owl (Tyto alba) and common wood pigeon 78 , and compound tubular type in wild bronze turkey (Meleagris gallopavo) 79 . The avian esophageal glands modify that have short ducts, and located in the lamina propria. While, mammalian esophageal glands are more developed dominated the submucosa 69 . The ducting system have some of characteristics different according animals species 73 . It is noted that esophageal mucous glands progressively develop with higher classes of the evolution tree. The geometric parameters of esophageal submucosal glands are measured in the avian and porcine species. The esophageal submucosal glands comprised 35% in avian and 45% in porcine area of the submucosa. The glands have an area of 125,000 μm 2 in avian) and 580,000 μm 2 in porcine 80 . Some fish esophagi lack the esophageal glands, instead mucous cells are located in the epithelium. This occurs in Sphoeroides testudineus 81 , Trichomycterus brasiliensis 82 and Anablepsoidesurophthalmus 74 , Nile tilapia and African catfish 75 , Larimichthys crocea (Acanthopterygii: Perciformes) 83 . While, other aquatic fish, the Asian seabass (Lates calcarifer), has both goblet cells and esophageal glands 84 . In crocodiles, the juvenile American alligators (Alligator mississippiensis), the esophageal epithelium has few mucous goblet cells 67 . The mucous secretion is essential to moisten food, lubrication and have protective function to neutralize the gastric acidity in case gastric reflux 85 . The evaluation of the stomach and the specification of gastric epithelium is described among fishes, amphibians, reptiles, birds and mammals 86 . Comparisons of stomach acidity in mammal and bird indicates that scavengers and carnivores exhibit higher stomach acidities compared to herbivores or carnivores feeding on phylogenetically distant prey 87 . Delicate submucosa is a characteristic feature of avian esophagus. This may be related to absence of the submucosal glands. unlike mammals, the abundant glandular subunits occupied the submucosa in dogs while extends to the tunica muscularis in rabbit 73 . www.nature.com/scientificreports/ The current study detected localization of collagen and reticular and elastic fibers in embryonic quail esophagus. Staining with Mallory trichrome and Weigert-Van Gieson revealed the presence of collagen fibers in the basal lamina, lamina propria, between the muscle fibers, and the serosa. Reticular fibers were detected in the lamina propria, myocytes, around glands, and in the myenteric or Auerbach's plexus. The observation of reticular fibers around the SMC was supported by previous studies 88 . Van-Geison stain was used to detect collagen in the pheasant 53 and geese 89 esophagus, particularly in the lamina propria and submucosa of the latter. The amount of collagen fibers in the avian esophagus depended on the feeding habits of the species. The lamina propria had more collagen fibers in pariah kites, egrets, and doves, while only a few collagen and reticular fibers were detected in goshawks and ducks. Collagen and elastic fibers are found in the tunica adventitia of the esophagus of pariah kites, median egrets, goshawks, doves, and ducks 61 .
In the current study, we used CD34, a common marker for undifferentiated stem cells, to demonstrate the distribution of different types of putative stem cells during the different stages of esophageal development. On the 5th day of incubation, CD34 positive cells were identified in the, subepithelial tissue, and mesenchyme, while on the 8th day they were also identified in the lamina propria and peri-muscular tissue. On the 15th day. The, interstitial cells including TCs and fibroblasts, exhibited CD34 immunoreactivity. Telocytes have an essential role in morphogenesis, embryogenesis, organization of the embryonic tissues [81][82][83][84][85][86][87][88][89][90][91][92][93][94][95][96][97] , cell migration, cell adhesion, proliferation, differentiation 33,35,96 , tissue homoeostasis, remodeling 98 and repair 99 . The CD34 protein is a member of a family of transmembrane sialomucin proteins. CD34 is common for marker of hematopoietic stem cells, hematopoietic progenitor cells, as well as non-hematopoietic cells such as vascular endothelial progenitors and embryonic fibroblasts, multipotent mesenchymal stromal cells, interstitial dendritic cells, and epithelial progenitors. CD34 is critical for adhesion molecules 100 , proliferation and blocking differentiation of stem or progenitor cells [87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] . www.nature.com/scientificreports/  www.nature.com/scientificreports/ Angiogenesis is the physiological process of forming new blood vessels from the pre-existing vessels. Growth of the vasculature is associated with proliferation of the endothelial cells to form sprouting vascular branches. Angiogenic events are essential during development to provide adequate nutrients for the microenvironment required by cells and tissues. If the oxygen levels are low, cells may experience hypoxia. Hypoxic cells express vascular proliferation markers to induce and enhance angiogenesis 104,105 . In the current study, we used immunohistochemistry to demonstrate the types of cells that express VEGF, a vascular proliferation marker. On the 5th day of incubation, VEGF positive cells were identified in the subepithelial tissue, and mesenchyme, and on the 8th day of incubation, they were also identified in the lamina propria and peri-muscular tissue. VEGF positive TCs were distinguished by their telopodes. On the 15th day of incubation, Telocytes may promote endothelial cell proliferation and angiogenesis 106 . Stromal fibroblasts express VEGF 107 . VEGF is secreted by the salivary gland and also shed in the saliva. Salivary VEGF is an essential stimulus for oral mucosal tissue repair 108,109 .

Conclusion
The current study provided information on esophageal development, including a timeline of the development events, and described changes of cellular components. The quail esophagus may be used as a model to study esophageal disorders. The current study also discussed the unique morphological features of quail esophagus and analyze the evolutionary morphology among different classes in relation to the function. www.nature.com/scientificreports/