Analysis of accessible chromatin landscape in the inner cell mass and trophectoderm of human blastocysts.

Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (<1 kb) and distal regions versus other regions were significantly different between ICM versus TE samples (P < 0.01). We detected that a higher percentage of accessible binding loci were located within 1 kb of the transcription start site in ICM compared to TE (P < 0.01). However, a higher percentage of accessible regions was detected in the distal region of TE compared to ICM (P < 0.01). In addition, eight differential peaks with a false discovery rate <0.05 between ICM and TE were detected. This is the first study to compare the landscape of the accessible chromatin between ICM and TE of human preimplantation embryos, which unveiled chromatin-level epigenetic regulation of cell lineage specification in early embryo development.

A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit.

During embryogenesis, differentiation of pluripotent cells into somatic cell types depends both on signaling cues and intrinsic gene expression programs. While the molecular underpinnings of pluripotency are well mapped, much less is known on how mouse embryonic stem cells (mESCs) differentiate. Using RNA-Seq profiling during specification to the three germ layers, we showed that mESCs switched on condition-specific gene expression programs from the onset of the differentiation procedure and that primed pluripotency did not constitute an obligatory intermediate state. After inferring the gene network controlling mESC differentiation, we tested the role of the highly connected nodes by deleting them in a triple knock-in Sox1-Brachyury-Eomes mESC line reporting on ectoderm, mesoderm, and endoderm fates. This led to the identification of regulators of mESC differentiation that acted at several levels: Sp1 as a global break on differentiation, Nr5a2 controlling ectoderm specification, and notably Fos:Jun and Zfp354c as opposite switches between ectoderm and mesendoderm fate.

Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?

OBJECTIVE: To investigate the blastocoelic fluid (BF) for the presence of DNA that could be amplified and analyzed; the extent to which its chromosomal status corresponds to that found in trophectoderm (TE) cells, polar bodies (PBs), or blastomeres; and the identification of segmental abnormalities. DESIGN: Longitudinal cohort study. SETTING: In vitro fertilization unit. PATIENT(S): Fifty-one couples undergoing preimplantation genetic screening or preimplantation genetic diagnosis for translocations by array-comparative genomic hybridization on PBs (n = 21) or blastomeres (n = 30). INTERVENTION(S): BFs and TE cells were retrieved from 116 blastocysts, whose chromosome status had already been established by PB or blastomere assessment. Separate chromosome analysis was performed in 70 BFs. MAIN OUTCOME MEASURE(S): Presence of DNA in BFs, evaluation of the chromosome condition, and comparison with the diagnosis made in TE cells and at earlier stage biopsies. RESULT(S): DNA detection was 82%, with a net improvement after refinement of the procedure. In 97.1% of BFs, the ploidy condition corresponded to that found in TE cells, with one false positive and one false negative. The rate of concordance per single chromosome was 98.4%. Ploidy and chromosome concordance with PBs were 94% and 97.9%, respectively; with blastomeres, the concordances were 95% and 97.7%, respectively. Segmental abnormalities, which were detected in PBs or blastomeres of 16 blastocysts, were also identified in the corresponding BFs. CONCLUSION(S): BF represents to a good extent the blastocyst ploidy condition and chromosome status when compared with TE cells. If the proportion of clinically useful BFs is improved, blastocentesis could become the preferred source of DNA for chromosomal testing.

Proteomic analysis of differences in ectoderm and mesoderm membranes by DiGE.

Ectoderm and mesoderm can be considered as prototypes for epithelial and mesenchymal cell types. These two embryonic tissues display clear differences in adhesive and motility properties, which are phenomenologically well characterized but remain largely unexplored at the molecular level. Because the key downstream regulations must occur at the plasma membrane and in the underlying actin cortical structures, we have set out to compare the protein content of membrane fractions from Xenopus ectoderm and mesoderm tissues using 2-dimensional difference gel electrophoresis (DiGE). We have thus identified several proteins that are enriched in one or the other tissues, including regulators of the cytoskeleton and of cell signaling. This study represents to our knowledge the first attempt to use proteomics specifically targeted to the membrane-cortex compartment of embryonic tissues. The identified components should help unraveling a variety of tissue-specific functions in the embryo.

The function of FGF signaling in the lens placode.

Previous studies suggested that FGF signaling is important for lens formation. However, the times at which FGFs act to promote lens formation, the FGFs that are involved, the cells that secrete them and the mechanisms by which FGF signaling may promote lens formation are not known. We found that transcripts encoding several FGF ligands and the four classical FGF receptors are detectable in the lens-forming ectoderm at the time of lens induction. Conditional deletion of Fgfr1 and Fgfr2 from this tissue resulted in the formation of small lens rudiments that soon degenerated. Lens placodes lacking Fgfr1 and 2 were thinner than in wild-type embryos. Deletion of Fgfr2 increased cell death from the initiation of placode formation and concurrent deletion of Fgfr1 enhanced this phenotype. Fgfr1/2 conditional knockout placode cells expressed lower levels of proteins known to be regulated by FGF receptor signaling, but proteins known to be important for lens formation were present at normal levels in the remaining placode cells, including the transcription factors Pax6, Sox2 and FoxE3 and the lens-preferred protein αA-crystallin. Previous studies identified a genetic interaction between BMP and FGF signaling in lens formation and conditional deletion of Bmpr1a caused increased cell death in the lens placode, resulting in the formation of smaller lenses. In the present study, conditional deletion of both Bmpr1a and Fgfr2 increased cell death beyond that seen in Fgfr2(CKO) placodes and prevented lens formation. These results suggest that the primary role of autocrine or paracrine FGF signaling is to provide essential survival signals to lens placode cells. Because apoptosis was already increased at the onset of placode formation in Fgfr1/2 conditional knockout placode cells, FGF signaling was functionally absent during the period of lens induction by the optic vesicle. Since the expression of proteins required for lens formation was not altered in the knockout placode cells, we can conclude that FGF signaling from the optic vesicle is not required for lens induction.

Proteomic analysis of the major cellular proteins of bovine trophectoderm cell lines derived from IVP, parthenogenetic and nuclear transfer embryos: Reduced expression of annexins I and II in nuclear transfer-derived cell lines.

Trophectoderm cell lines were established from 8-day in vitro-cultured embryos of cattle derived from fertilization (IVF), somatic cell nuclear transfer (NT), or parthenogenetic activation (P) of in vitro-matured oocytes and from five 8-day-old in vivo (V) embryos. The most abundant cellular proteins of 5 V-, 16 NT-, 12 P-, and 16 IVF-derived cell lines were compared by 2D-gel electrophoresis and mass spectrometry; that is, the unaltered thiourea/urea extract of each cell culture was analyzed. Common protein spots (n=118) were examined, and 95% were identified with significant scores from protein and gene database searches. Of the proteins detected and identified, actin and cytokeratin-8 were found to be the most abundant. Other prominent cellular proteins were metabolic enzymes such as aldose reductase, phosphoglycerate mutase, enolase, triosephosphate isomerase, cytoskeletal interacting proteins transgelin and stratifin, anti-oxidant proteins peroxiredoxin 1 and anti-oxidant protein 2, and the calcium-dependent lipid-binding proteins annexins I and II. In comparative analysis of the 2D-gels, the NT-derived trophectoderm had less annexins I and II in comparison to the IVF- and P-derived trophectoderm. Because annexins I and II are abundant in the placenta and have functions important to the maintenance of placentation, the down-regulation of the annexin genes in the cultured NT trophectoderm may be related to the frequent failures of NT pregnancies.

Expression of Wise in chick embryos.

We have performed in situ hybridization to study the expression of Wise in early chick embryos. Wise expression is first detectable in the ectoderm at posterior levels of late neurula. As development proceeds, Wise expression is seen in specific patterns in the ectoderm of the trunk region, pharyngeal arches, limb buds, and feather buds. In addition to these areas, particular cartilages such as the ones in the maxillary process and limbs start to express Wise at the late pharyngula stage, and the expression in these cartilages becomes stronger than that in epidermal components at later stages. Importantly, Wise is expressed in regions where other signaling molecules such as Wnt, Bmp, and Shh are known to function in morphogenesis and differentiation. Direct comparisons of the expression of Wise and these genes are also demonstrated.

Negative regulation of Activin/Nodal signaling by SRF during Xenopus gastrulation.

Activin/Nodal signaling is essential for germ-layer formation and axial patterning during embryogenesis. Recent evidence has demonstrated that the intra- or extracellular inhibition of this signaling is crucial for ectoderm specification and correct positioning of mesoderm and endoderm. Here, we analyzed the function of Xenopus serum response factor (XSRF) in establishing germ layers during early development. XSRF transcripts are restricted to the animal pole ectoderm in Xenopus early embryos. Ectopic expression of XSRF RNA suppresses mesoderm induction, both in the marginal zone in vivo and caused by Activin/Nodal signals in animal caps. Dominant-negative mutant or antisense morpholino oligonucleotide-mediated inhibition of XSRF function expands the expression of mesendodermal genes toward the ectodermal territory and enhances the inducing activity of the Activin signal. SRF interacts with Smad2 and FAST-1, and inhibits the formation of the Smad2-FAST-1 complex induced by Activin. These results suggest that XSRF might act to ensure proper mesoderm induction in the appropriate region by inhibiting the mesoderm-inducing signals during early embryogenesis.

An essential role for zebrafish Fgfrl1 during gill cartilage development.

The vertebrate craniofacial skeleton develops via a complex process involving signaling cascades in all three germ layers. Fibroblast growth factor (FGF) signaling is essential for several steps in pharyngeal arch development. In zebrafish, Fgf3 and Fgf8 in the mesoderm and hindbrain have an early role to pattern the pouch endoderm, influencing craniofacial integrity. Endodermal FGF signaling is required for the differentiation and survival of postmigratory neural crest cells that form the pharyngeal skeleton. We identify a novel role for zebrafish Fgf receptor-like 1a (Fgfrl1a) that is indispensable during gill cartilage development. We show that depletion of Fgfrl1a is sufficient to abolish cartilage derivatives of the ceratobranchials. Using an Fgfrl1a-deficient model, we analyzed expression of genes critical for chondrogenesis in the different compartments of the developing pharyngeal arch. Fgfrl1a-depleted animals demonstrate typical neural crest specification and migration to populate the arch primordia as well as normal pouch segmentation. However, in the absence of Fgfrl1a, larvae fail to express the transcription factor glial cells missing 2 (gcm2), a gene necessary for cartilage and gill filament formation, in the ectodermal lining of the branchial arches. In addition, two transcription factors essential for chondrogenesis, sox9a and runx2b, fail to express within the mesenchymal condensations of the branchial arches. A duplicate zebrafish gene, fgfrl1b, has now been identified. We show that Fgfrl1b is also required for proper formation of all ventral cartilage elements and acts cooperatively with Fgfrl1a during gill cartilage formation.

Sp5l is a mediator of Fgf signals in anteroposterior patterning of the neuroectoderm in zebrafish embryo.

The neuroectoderm is patterned along the anterior-posterior axis in vertebrate embryos. Fgf signals are required to induce the posterior neuroectodermal fates, but they repress the anterior fate. Sp5l/Spr2, an Sp1-like transcription factor family member, has been shown to be required for development of mesoderm and posterior neuroectoderm. We demonstrate here that repression of the anterior neuroectodermal markers fez and otx1 by fgf17b or fgf3 coincides with induction of sp5l in the anterior neuroectoderm, and that this repression is efficiently rescued by simultaneous sp5l knockdown. On the other hand, sp5l knockdown is able to inhibit inductive activity of ectopic Fgf signals on the expression of the posterior neuroectodermal markers gbx2, hoxb1b, and krox20. Furthermore, effect of overexpression of a dominant negative Fgf receptor on anteroposterior patterning of the neuroectoderm is rescued by sp5l overexpression. Taken together, these data suggest that sp5l mediates the functions of Fgf signals in anteroposterior patterning of the neuroectoderm during zebrafish embryogenesis.

Basement membrane heterogeneity during chick development as shown by tomato (Lycopersicon esculentum) lectin binding.

Basement membranes (BMs) constitute a distinct compartment of the extracellular matrix (ECM). All BMs show a similar structural appearance but differ in molecular composition. These variations have critical functional implications. The aim of this study is to establish the pattern of the tomato lectin (Lycopersicon esculentum agglutinin--LEA) binding sites in the BMs of the developing chick embryo (stages 4-21, Hamburger and Hamilton, 1951) in order to achieve a better understanding of the molecular heterogeneity of BMs. The study was performed with transmission electron microscopy (TEM) histochemistry, and confocal laser microscopy. TEM showed that LEA bound to the lamina densa and to the lamina fibroreticularis of the BMs. Through the period studied, most of the LEA binding appeared in the ectodermal BM and its derivatives. In the limb bud, LEA binding to the ectoderm BM was more intense in the ventral half than in the dorsal half. Furthermore, LEA allowed the early (HH16) detection of the transverse fibrillar tracts. In the lens and in the inner ear primordium, the BMs were LEA positive through the placode and cup stages. The binding was progressively reduced through the vesicle stage. The BMs of the olfactory primordium, and of the Rathke's pouch were positive. In contrast, the BMs of the developing central nervous system were negative. The BMs of both the paraxial and the lateral plates of the mesoderm were negative, whereas the notochord and the BM of the Wolffian duct were positive. The endodermal BM and its derivatives were negative. The ECM located between the fusing endocardial tubes, and the BM of the fusion zone of the paired aortae, were positive. This suggested an active role of the LEA-positive glycoproteins in the fusion of endothelia. Our results show the heterogeneity of the chick embryo BMs during development. In addition, LEA constitutes an excellent marker for the primordial germ cells.

Regulatory targets for transcription factor AP2 in Xenopus embryos.

The transcription factor AP2 (TFAP2) has an important role in regulating gene expression in both epidermis and neural crest cells. In order to further characterize these functions we have used a hormone inducible TFAP2alpha fusion protein in a Xenopus animal cap assay to identify downstream targets of this factor. The most common pattern comprised genes predominantly expressed in the epidermis. A second group was expressed at high levels in the neural crest, but all of these were also expressed in the epidermis as well as in other tissues in which TFAP2alpha has not been detected, suggesting modular control involving both TFAP2-dependent and TFAP2-independent components. In addition, a few strongly induced genes did not overlap at all in expression pattern with that of TFAP2alpha in the early embryo, and were also activated precociously in the experimentally manipulated ectoderm, and thus likely represent inappropriate regulatory interactions. A final group was identified that were repressed by TFAP2alpha and were expressed in the neural plate. These results provide further support for the importance of TFAP2alpha in ectoderm development, and also highlight the molecular linkage between the epidermis and neural crest in the Xenopus embryo.

An essential role of Xenopus Foxi1a for ventral specification of the cephalic ectoderm during gastrulation.

During gastrulation in Xenopus, the head ectoderm is subdivided into the central nervous system (CNS) anlage (neural plate) and the non-CNS ectoderm (i.e. epidermis, placodes and neural crest). The winged-helix transcription factor Xfoxi1a is one of the earliest markers for the preplacodal region at the mid-neurula stage. Interestingly, before the establishment of the preplacodal region, Xfoxi1a expression is detected in the entire cephalic non-neural ectoderm at the mid- and late gastrula stages. The present study focuses on the role of Xfoxi1a particularly at the gastrula stages. The early Xfoxi1a expression in the anteroventral ectoderm is dependent on Bmp signals and suppressed by Wnt signals. Inhibition of Xfoxi1a activities by injection of antisense oligonucleotides leads to suppression of non-CNS ectodermal markers (e.g. keratin) and expansion of the anterior expression domain of the CNS marker Sox2. Conversely, misexpression of Xfoxi1a suppresses Sox2 and induces keratin in the anterior neural plate. In the animal cap, Xfoxi1a overexpression antagonizes the neuralizing activity of Chordin (Chd). Studies using an inducible Xfoxi1a construct (GR-Xfoxi1a) show that the ventralizing function of Xfoxi1a is confined to the gastrula stage. Thus, Xfoxi1a is an essential regulator of ventral specification of the early head ectoderm during gastrulation.

In vitro attachment of bovine hatched blastocysts on fibronectin is mediated by integrin in a RGD dependent manner.

We investigated the effect of extracellular matrix protein on in vitro attachment and outgrowth of bovine hatched blastocysts. In vitro produced bovine hatched blastocysts were cultured on a fibronectin- or laminin-coated Petri dishes. Hatched blastocysts adhered and outgrew on the fibronectin-coated dish whereas no attachment was observed on the laminin-coated dish. The attachment and outgrowth on fibronectin were significantly inhibited in the presence of synthetic peptides containing the Arg-Gly-Asp (RGD) sequence, which interacts with the fibronectin receptor (integrin alpha5beta1), but were not inhibited by the control peptides containing the Arg-Gly-Glu (RGE) sequence. Addition of anti-fibronectin receptor (integrin alpha5beta1) antibody to the culture medium also inhibited the attachment and outgrowth on fibronectin-coated Petri dishes. Subsequently we examined mRNA expression and protein expression of alpha5 and beta1 integrin subunit in the hatched blastocyst by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. Expression of both mRNA and protein were detected in blastocysts. These results indicate that trophectoderm cells of bovine hatched blastocysts have already acquired the ability to adhere and outgrow on fibronectin in vitro by an integrin- mediated manner.

High-throughput functional screen of mouse gastrula cDNA libraries reveals new components of endoderm and mesoderm specification.

This study describes a cross-species functional screen of mouse gastrula cDNA libraries for components of endoderm and mesoderm specification. Pools of 96 cDNAs from arrayed mouse gastrula cDNA libraries were transcribed into mRNA and injected into either the presumptive mesoderm or the ectoderm of one-cell Xenopus laevis embryos. Injected embryos were examined at gastrula stage by in situ hybridization with endoderm or mesoderm markers. Using this approach, we screened over 700 pools or approximately 60,000 cDNAs. We identified 17 unique cDNAs that function during mesoderm and/or endoderm specification and 16 that cause general morphology changes. Identified molecules fall into eight general functional groups as follows: cell cycle components (seven), transcription factors (four), extracellular secreted molecules (seven), transmembrane receptors (one), intracellular signaling components (five), microtubule components (two), metabolism molecules (three), and unknown (four). Several of the genes we identified would not have been predicted to be involved in endoderm or mesoderm specification, highlighting the usefulness of nonbiased screening approaches. This includes Otx2, which we show is a downstream target of Xsox17beta. The speed, low cost, and high efficiency of this cross-species screen makes it an ideal method for examining cDNAs from difficult-to-obtain sources. Therefore, this approach complements the current mouse molecular genetics systems and provides a powerful means for the genome-wide examination of mammalian gene function.

Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification.

We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.

Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells.

Based on evidence suggesting similarities to human embryonic stem cells, human embryonic germ (hEG) cells have been advocated as an alternative pluripotent stem cell resource but have so far received limited attention. To redress this imbalance, human fetal gonads were collected for the isolation and culture of primordial germ cells at 7-9 weeks postconception. We provide evidence for the derivation, culture, and differentiation of hEG cells in vitro. This evidence includes the expression of markers characteristic of pluripotent cells, the retention of normal XX or XY karyotypes, and the demonstration of pluripotency, as suggested by the expression of markers indicative of differentiation along the three germ lineages (ectoderm, mesoderm, and endoderm) and an associated loss of pluripotent markers. In assessing this differentiation, however, we also demonstrate a hitherto unacknowledged overlap in gene expression profiles between undifferentiated and differentiated cell types, highlighting the difficulty in ascribing cell lineage by gene expression analyses. Furthermore, we draw attention to the problems inherent in the management of these cells in prolonged culture, chiefly the difficulty in preventing spontaneous differentiation, which hinders the isolation of pure, undifferentiated clonal lines. While these data advocate the pursuit of pluripotent hEG cell studies with relevance to early human embryonic development, culture limitations carry implications for their potential applicability to ambitious cell replacement therapies.

Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos.

The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the tropho-blast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.

Ber-EP4 immunoreactivity depends on the germ layer origin and maturity of the squamous epithelium.

AIM: To map the expression of Ber-EP4 in well-differentiated squamous epithelia, metaplastic squamous epithelia and dysplastic squamous epithelia of different origins. METHODS AND RESULTS: Squamous epithelium of different origin was stained using a standard immunohistochemistry method applied to paraffin sections. We found that normal squamous epithelium of the oral cavity, oesophagus, uterine cervix, vagina, anal canal, and branchial cysts are Ber-Ep4-negative, as are the mature squamous metaplasia of bronchial mucosa, urinary bladder mucosa and uterine cervical mucosa. In contrast, immature squamous metaplasia of bronchial mucosa, or uterine cervical mucosa, and squamous dysplasia of oral mucosa of endodermal origin, or uterine cervical mucosa in most cases expressed Ber-EP4. CONCLUSION: Squamous epithelia of ectodermal origin never express Ber-EP4, whether normal, hyperplastic, dysplastic or neoplastic. In contrast, squamous epithelium of endodermal origin sometimes contains the target glycoproteins of Ber-EP4 when immature, metaplastic, dysplastic or neoplastic. The results indicate that the differences in expression of Ber-EP4 in squamous epithelium depend primarily on germ layer origin, and on the maturity of the epithelium.