Lineage tracing of the endoderm during oral development

Background: The contribution of the endoderm to the oral tissues of the head has been debated for many years. With the arrival of Cre/LoxP technology endoderm progenitor cells can now be genetically labeled and tissues derived from the endoderm traced. Using Sox17-2A-iCre/Rosa26 reporter mice we have followed the fate of the endoderm in the teeth, glands, and taste papillae of the oral cavity. Results: No contribution of the endoderm was observed at any stage of tooth development, or in development of the major salivary glands, in the reporter mouse during development. In contrast, the minor mucous glands of the tongue were found to be of endodermal origin, along with the circumvallate papilla and foliate papillae. The mucous minor salivary glands of the palate, however, were of mixed ectodermal and endodermal origin. Conclusions: In contrast to urodele studies, the epithelium of murine teeth is derived solely from the ectoderm. The border between the ectoderm- and endoderm-derived epithelium may play a role in determining the position of the lingual glands and taste buds, and may explain differences observed between taste buds in the anterior and posterior part of the tongue.     

Bmp signaling is necessary and sufficient for ventrolateral endoderm specification in Xenopus

Here we show that Bmp signaling is necessary and sufficient for the specification of ventral endoderm in Xenopus embryos. Overexpression of Bmp4 in ectoderm induces markers of endoderm, including Sox17beta, Mixer, and VegT, but cannot induce the expression of the dorsoanterior markers, Xhex and Cerberus. Furthermore, knockdown approaches using overexpression of Bmp antagonists and morpholinos designed against Bmp4, Bmp2, and Bmp7 demonstrate that Bmp signaling is critical for ventral, but not dorsoanterior endoderm formation. This activity is not simply a result of embryonic dorsalization as markers for dorsal endoderm are not expanded. We further show that endodermal cells of either ventral or dorsal character do not form when both Wnt and Bmp signals are abolished. Overall, this report strongly suggests that Bmp plays an essential role in ventral endoderm specification.     

Dual lineage-specific expression of sox17 during mouse embryogenesis

Sox17 is essential for both endoderm development and fetal hematopoietic stem cell (HSC) maintenance. While endoderm-derived organs are well known to originate from Sox17-expressing cells, it is less certain whether fetal HSCs also originate from Sox17-expressing cells. By generating a Sox17(GFPCre) allele and using it to assess the fate of Sox17-expressing cells during embryogenesis, we confirmed that both endodermal and a part of definitive hematopoietic cells are derived from Sox17-positive cells. Prior to E9.5, the expression of Sox17 is restricted to the endoderm lineage. However, at E9.5 Sox17 is expressed in the endothelial cells (ECs) at the para-aortic splanchnopleural region that contribute to the formation of HSCs at a later stage. The identification of two distinct progenitor cell populations that express Sox17 at E9.5 was confirmed using fluorescence-activated cell sorting together with RNA-Seq to determine the gene expression profiles of the two cell populations. Interestingly, this analysis revealed differences in the RNA processing of the Sox17 mRNA during embryogenesis. Taken together, these results indicate that Sox17 is expressed in progenitor cells derived from two different germ layers, further demonstrating the complex expression pattern of this gene and suggesting caution when using Sox17 as a lineage-specific marker. STEM Cells2012;30:2297-2308.     

Indirect immunofluorescent staining of fibronectin associated with the floor of the foregut during formation and rupture of the oral membrane in the chick embryo

The distribution of the glycoprotein, fibronectin, within the cranial region of stage 8–16 chick embryos was examined by indirect immunofluorescence using paraffin sections exposed to affinity-purified rabbit anti-human CIG and FITC-conjugated goat anti-rabbit immunoglobulins. Fluorescence was present within the matrix surrounding the cranial mesenchyme, along the basal surfaces of all epithelia, and surrounding the notochord at all stages. Fluorescence associated with the floor of the foregut was particularly intense. The fluorescent layers beneath the ectoderm and endoderm of the oral (oropharyngeal) membrane at stage 8 merged into a single, continuous, intensely fluorescent line as the extra-cellular space within the oral membrane narrowed during stages 9–12. This line of uniform fluorescence parallels the previously described histological reorganization of the extracellular compartment of the oral membrane, but the ultrastructural localization of this fluorescent material remains unknown. Fluorescence was also intense beneath the foregut endoderm in the presumptive cardiac region caudal to the oral membrane and was continuous with strands of fluorescent material extending into the matrix of the dorsal mesocardium and cardiac jelly of the developing tubular heart. These observations indicate that the extracellular matrix associated with the floor of the entire foregut contains fibronectin during stages encompassing the formation and rupture of the oral membrane. The presence of fibronectin within the oral membrane and dorsal mesocardium, as well as between Rathke's pouch and infundibulum and within the closing plates between ectodermal clefts and endodermal pouches, is consistent with the possibility that this glycoprotein may play a role in adhesion at these sites.     

Endodermal/ectodermal interfaces during pharyngeal segmentation in vertebrates

A key event in the formation of the pharyngeal arches is the outpocketing of the endodermal pharyngeal pouches and the establishment of contact with the overlying ectoderm. However, relatively little is known about how the endoderm and ectoderm relate to each other at these points of contact and the extent to which this differs between the pouches. We have therefore detailed the interactions between the pharyngeal pouches and ectoderm in the chick embryo. Unlike the other pouches, the first pouch does not sustain direct contact with the ectoderm but separates after initial contact. Contrastingly, a perforation is formed between the second pouch and cleft that creates an external opening into the pharynx. Finally, the third and fourth pouch endoderm can be seen to bulge outwards through the ectoderm, although external openings to the pharyngeal lumen are not established. To understand whether these behaviours represent derived or ancestral features, we characterised the pharyngeal ectodermal–endodermal interfaces in the shark embryo. We found that the pouches of the posterior gill-bearing arches in this species also displayed the outward bulging of the endoderm into the ectoderm, although openings were established. We further used genetic tools to detail unambiguously the relationship between the endoderm and ectoderm in zebrafish and mouse embryos and again found that the posterior pouches break through the ectoderm. Thus different pharyngeal pouches establish different topological relationships with the overlying ectoderm and the posterior pouches initiate the developmental programme for the formation of gills, be they amniotes or anamniotes.     

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human‐like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal–epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two‐step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron‐dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam‐like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid‐somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations.     

Developmental processes and ectodermal contribution to the anal canal in mice

The anorectal canal has two origins; the upper part is derived from endoderm and the lower part is derived from ectoderm. The process of ectodermal contribution to the canal remains unclear. To understand the development of this area, serial sagittal sections of mouse embryos were made every 12 h from embryonic day 13.0 (E13.0) to E18.5. Three-dimensional (3-D) reconstructions were obtained from these sections. At the time of the disappearance of the cloacal membrane (E13.5), the endodermal lining reached the site of disintegrated membrane. Thus, the whole canal was of endodermal origin. The transitional zone between the dorsal end of the primary perineum and tail was thicker than other ectodermal epithelia. In this region, it changed from an acute to obtuse angle. After it straightened out and formed the canal, the secondary perineum appeared caudally. During these processes, the external sphincter appeared in the underlying mesenchyme of the thick ectoderm and functioned as a drawstring to form the ectodermal anal canal.     

The intermediate filament cytoskeleton of malignant mesotheliomas and its diagnostic significance

The intermediate filament cytoskeleton of epithelial, biphasic, and fibrous malignant pleural mesotheliomas was studied by immunohistochemistry and gel electrophoresis. The results were compared with data similarly obtained from lung adenocarcinomas. All mesotheliomas immunostained with various monoclonal and polyclonal antibodies against cytokeratins. By double immunofluorescence microscopy, coexpression of cytokeratins and vimentin was found in the fusiform cells of biphasic and fibrous mesotheliomas. As determined by two-dimensional gel electrophoresis, lung adenocarcinomas exclusively expressed Cytokeratins 7, 8, 18, and 19, and the same polypeptides were found in the fibrous mesotheliomas. These four cytokeratins were also found in the epithelial and biphasic mesotheliomas, most of which, however, also expressed, additional cytokeratins, such as the basic Polypeptide 5 and, in some cases, Cytokeratins 4, 6, 14, and 17. The results demonstrate the epithelial nature of all types of malignant mesotheliomas and thus justify their classification as carcinomas. When epithelial morphology is evident, the pattern of cytokeratin expression is usually more complex, as indicated by the synthesis, in addition to the "simple epithelial" pattern (7, 8, 18, and 19), of certain cytokeratin polypeptides which hitherto have been presumed to be typical of stratified epithelia. This cytokeratin complexity and the coexpression of vimentin and cytokeratins in certain forms of mesotheliomas indicate that these tumors are a clearly distinct and complex group of carcinomas. Their special cytoskeletal filament protein expression should prove useful in differentiating mesotheliomas from other carcinomas, particularly from adenocarcinomas growing in the lung.     

Distribution of fibronectins and laminin in the early pig embryo

Fibronectins (FN) and laminin (LN) distributions were studied in the pig embryo by indirect immunofluorescence using antiporcine FN and antimurine LN antibodies. Extracellular FN are first detected in the early blastocyst before endodermal cell migration. They appear between the cells and on the blastocoelic face of the inner cell mass; thus, they are located at the interface of the trophectoderm and extraembryonic endoderm. Mesodermal cells migrate in a tridimensional network of fibrillar FN. These glycoproteins are also in the extraembryonic membranes (chorion and yolk sac wall) contiguous to the FN-rich basement membranes of embryonic ectoderm and endoderm. Extracellular LN appears in the blastocyst when the endoderm is already established as a continuous cellular monolayer, and is located between the trophectoderm and the extraembryonic endoderm, which produces it. Laminin also accumulates at the basal surface of the embryonic ectoderm at the onset of gastrulation. In the extraembryonic membranes, LN appears at the interface of the endoderm and mesoderm and at the interface of the trophectoderm and mesoderm. It is produced and secreted by extraembryonic mesodermal cells. Analysis of the distribution of these glycoproteins suggests that FN allow the migration of endodermal and mesodermal cells by providing them with a suitable substrate. When these cells become immobilized, they synthesize LN, probably to stabilize their interactions with the underlying extracellular material and epithelia.     

The histogenetic-embryologic basis for reappearance of alpha-fetoprotein in endodermal sinus tumors (yolk sac tumors) and teratomas.

The mechanism of neosynthesis of the human tumor-associated fetal antigen alpha-fetoprotein (AFP) in a variable percentage of patients with testicular, ovarian and extragonadal germ cell tumors has generally been considered unknown or beyond any simple explanation. Of decisive importance is the cellular basis for AFP production 1. in ontogenesis and 2. in malignancy as dependent on an exact tumor histogenesis. Based on (1) the histogenetic-embryologic classification of germ cell tumors and the concept of yolk sac tumor (or endodermal sinus tumor), (2) the available clinical and experimental observations, and (3) the immunofluorescent localization of AFP in the endodermal sinus tumor of the human testis, it is concluded that AFP synthesis in these neoplasms is explained by the fact that they contain yolk sac endoderm, which produce AFP analogous with the physiological AFP synthesis by the fetal yolk sac in early embryogenesis.     

小鼠iPS细胞体外自发性分化及神经定向分化中基因表达谱的研究

目的:研究小鼠诱导多能干细胞(i PS细胞)在体外自发性分化和神经定向分化过程中的基因表达谱的特性。方法:在体外将i PS细胞分别进行形成类胚胎小体后自发性分化及成骨蛋白抑制剂Noggin诱导神经定向分化后,通过实时荧光定量PCR(q PCR)测定在分化过程中i PS细胞分化相关基因表达的变化。结果:i PS细胞形成类胚胎小体后胚胎外胚层标志基因(GFAP,Map2和Tu J1)及内胚层标记基因(Foxa2,GATA4和Sox17)的表达随分化而迅速增加,但中胚层标记基因(Bmp4,BRA,FGF5)表达水平变化不明显。在神经定向分化过程中,i PS细胞的神经标志物基因(Map2,Neu N,Tu J1和Sox1)及线粒体相关基因(12s,ND3,ND5,ND6、Cytb和Cox1)的表达都逐渐增加,且两者具有相同的增长趋势。结论:在自发性分化过程中小鼠i PS细胞具有自发向外胚层和内胚层分化的趋势;在神经定向分化过程中,i PS细胞的线粒体相关基因表达随着分化表达逐渐增加,并与神经细胞标记基因具有正相关性,表明线粒体的功能在i PS细胞神经定向分化中发挥重要作用。     

Notch signaling can regulate endoderm formation in zebrafish.

Early in vertebrate development, the processes of gastrulation lead to the formation of the three germ layers: ectoderm, mesoderm, and endoderm. The mechanisms leading to the segregation of the endoderm and mesoderm are not well understood. In mid-blastula stage zebrafish embryos, single marginal cells can give rise to both endoderm and mesoderm (reviewed by Warga and Stainier [2002] The guts of endoderm formation. In: Solnica-Krezel L, editor. Pattern formation in zebrafish. Berlin: Springer-Verlag. p 28-47). By the late blastula stage, however, single marginal cells generally give rise to either endoderm or mesoderm. To investigate this segregation of the blastoderm into cells with either endodermal or mesodermal fates, we analyzed the role of Notch signaling in this process. We show that deltaC, deltaD, and notch1 are expressed in the marginal domain of blastula stage embryos and that this expression is dependent on Nodal signaling. Activation of Notch signaling from an early stage leads to a reduction of endodermal cells, as assessed by sox17 and foxA2 expression. We further find that this reduction in endoderm formation by the activation of Notch signaling is preceded by a reduction in the expression of bonnie and clyde (bon) and faust/gata5, two genes necessary for endoderm formation (Reiter et al. [1999] Genes Dev 13:2983-2995; Reiter et al. [2001] Development 128:125-135; Kikuchi et al. [2001] Genes Dev 14:1279-1289). However, activation of Notch signaling in bon mutant embryos leads to a further reduction in endodermal cells, also arguing for a bon-independent role for Notch signaling in endoderm formation. Altogether, these results suggest that Notch signaling plays a role in the formation of the endoderm, possibly in its segregation from the mesoderm.     

A molecular signature for purified definitive endoderm guides differentiation and isolation of endoderm from mouse and human embryonic stem cells

Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular "definition" of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP? progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.     

Immunofluorescent Demonstration of Alpha-fetoprotein in Tissue Culture from Human Endodermal Sinus Tumors, Rat Yolk Sac Carcinoma and Fetal Rat Yolk Sac

An in vitro model for study of AFP (alpha-fetoprotein) synthesis is presented using immunofluorescent microscopic examination of cultured cells from rat yolk sac, experimental rat yolk sac carcinoma and human endodermal sinus tumors (EST).
The presence of positive staining for AFP in all cases by use of specific rabbit antisera against rat and human AFP supports the concept that AFP synthesis in germ cell tumors is confined to the yolk sac endoderm. The variation of positive staining cells due to a variable protein synthesis is discussed.     

Development of thymus,parathyroids, and ultimo-branchial bodies in NMRI and nude mice

In the NMRI mouse embryo, the thymus develops from the third endodermal pouch and the third ectodermal cleft. The cervical vesicle, formed not by the closure of the sinus cervicalis but by an invagination of the dorsal segment of the ectoderm between the third and fourth branchial clefts, contributes to the formation of the thymus. The intense proliferation of the ectoderm of the third cleft on the eleventh day covers the endodermal part. The thymus is thus composed of a central endodermal region and a peripheral ectodermal region. The normal adult thymus, then, has a mixed origin, the cortical cells being ectodermal, and the medullary cells endodermal in origin. The fourth endodermal pouch gives rise to the ultimo-branchial body, which becomes entirely incorporated within the thyroid on the fourteenth day. There is no formation of a thymus IV nor of a second pair of parathyroids from the fourth endodermal pouch. In the Nude mouse embryo, the third branchial pouch and cleft, as well as the cervical vesicle, develop normally for the first 11½ days. From this point on, the ectoderm of the third cleft ceases to develop further. The endoderm is, now, no longer covered by ectoderm and, deprived of its normal inducing agent, ceases to develop further. Thymic dysgenesis is thus ectodermal in origin. The fourth endodermal pouch develops normally. No development of an accessory “thymus-like” structure from the fourth pouch was observed. The dysgenetic thymus originates entirely from the third branchial pouches and clefts. In both Nude and NMRI embryos, the parathyroids develop at about 11½ days from a very limited area in the dorsal region of the cranial wall of the third endodermal pouch between the pharyngo-branchial and ecto-branchial ducts. Morphometric analysis shows that the volume of the parathyroids is the same in both strains of mice at each stage of development; nor does their microscopic appearance differ. Thus, mutation in the Nude mouse does not affect the development of the parathyroids from the third pouch, even though the first anomalies in the development of the thymus are observed at the precise moment at which the parathyroid primordium appears.     

Scale and tooth phenotypes in medaka with a mutated ectodysplasin-A receptor: implications for the evolutionary origin of oral and pharyngeal teeth

Ectodermal contribution to the induction of pharyngeal teeth that form in the endodermal territory of the oropharyngeal cavity in some teleost fishes has been a matter of considerable debate. To determine the role of ectodermal cell signaling in scale and tooth formation and thereby to gain insights in evolutionary origin of teeth, we analyzed scales and teeth in rs-3 medaka mutants characterized by reduced scale numbers due to aberrant splicing of the ectodysplasin-A receptor (edar). Current data show that, in addition to a loss of scales (83% reduction), a drastic loss of teeth occurred in both oral (43.5% reduction) and pharyngeal (73.5% reduction) dentitions in rs-3. The remaining scales of rs-3 were irregular in shape and nearly 3 times larger in size relative to those of the wild-type. In contrast, there was no abnormality in size and shape in the remaining teeth of rs-3. In wild-type medaka embryos, there was a direct contact between the surface ectoderm and rostral endoderm in pharyngeal regions before the onset of pharyngeal tooth formation. However, there was no sign of ectodermal cell migration in the pharyngeal endoderm and hence no direct evidence of any ectodermal contribution to pharyngeal odontogenesis. These data suggest differential roles for Eda-Edar signaling in the induction and growth of scales and teeth and support the intrinsic odontogenic competence of the rostral endoderm in medaka.     

P19 Cells Overexpressing Lhx1 Differentiate into the Definitive Endoderm by Recapitulating an Embryonic Developmental Pathway

Background

Epiblasts occur at the last pluripotent stage of embryonic development and are important in elucidating how the three germ layers are formed. However, little is known of the molecular mechanisms of their development. We have shown that LIM homeobox 1 (Lhx1) was involved in epiblast development in embryonic stem cells, especially meso- and endodermal differentiation. However, since epiblasts in embryoid bodies spontaneously develop into a further stage, it is difficult to study their development in this system.

Methods

Mouse embryonal carcinoma P19 cells which have properties similar to those of epiblasts provided new avenues of investigation into the regulatory mechanism of epiblasts.

Results

Overexpression of Lhx1 in P19 cells induced expression of organizer marker genes (Cer1, Gsc) and endoderm marker genes (Gata6, Foxa2, Sox17) but not extra-embryonic endoderm marker genes (Sox7 or Hnf4alpha).

Conclusion

This study suggested that Lhx1 overexpression caused P19 cells to differentiate into an endodermal lineage. Thus, P19 cells and their derivatives can be a useful model system to study how the three germ layers are formed.