Bilaminar Research Articles

Basal delamination during mouse gastrulation primes pluripotent cells for differentiation

The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and invivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.

Poster Session II: Development of transparency in the human lens cells.

As the neural tube forms in the fifth week of human embryonic development, a few ectodermal cells adjacent to the neural plate in the trilaminar embryo, swell, thicken, and compress their intercellular space to form a minute lens placode. At this early stage, the embryonic cells in the lens placode resemble neuroectoderm, enriched in cytoskeletal proteins, junctions, membrane channels, and stress response proteins. With continued differentiation, intracellular organelles disappear to decrease light scattering. The expression of high concentrations of crystallin proteins increase the index of refraction, "n", for optimal optical function. In each cell, the condensed cytoplasmic proteins organize into the short-range order necessary for maximal transmission of light. Actin, intermediate filaments and microtubules provide a scaffold for the organization of connexins, and aquaporin channels in the membranes supporting a unique microcirculation for the symmetric transport of fluid, ions, and nutrients to the layers of elongating, close-packed, hexagonal cells, the basis for this unique, biological, optical element. The entire optical system is stabilized by molecular and cellular mechanisms designed to protect optical function for a lifetime. Even with the specialized protective mechanisms in lens cells, loss of transparency in cataract formation is one of the most common effects of cellular aging accounting for over 50% of vision impairment worldwide.

Embryology – The Story of Us: Part 2

In the first part of this Anatomy and Physiology in Focus series (see: The Student Midwife, July 2021), we followed the development of the embryo from the point of fertilisation until the formation of the trilaminar disc. At this point, the three germ layers of the human body have formed during the process called gastrulation: the ectoderm, mesoderm, and endoderm. In this second part, we shall follow the development of the embryo from the beginning of the third week until the end of the embryonic period.

Application of dural bilaminar saturation in frontal-temporal skull defect repairs after standard decompressive craniectomy

Objective To approach the operative effect of dural bilaminar saturation in frontal-temporal skull defect repairs after standard decompressive craniectomy. Methods Fifty-two patients with titanium cranioplasty in frontal -temporal skull defect were retrospectively analyzed;27 of them adopted dural bilaminar saturation in the first standard decompressive craniectomy(test group);25 of them adopted dural monostratal saturation in the first operation(control group). The operation time,blood loss, postoperative complications in the two groups were compared. Results The mean operation time: the test group was(65.00±8.15)min and the control group was(83.00±10.25) min, there was statistically significant differences between the two groups(t=7.059,P 0.05). Conclusion Skull repairs in second standard decompressive craniectomy by dural bilaminar saturation, can be shortened the operation time and lessened amount of bleeding. Key words: Skull defects; Dural bilaminar saturation; Standard decompressive craniectomy

Peritoneal carcinomatosis: A malignant disease with an embryological origin?

IntroductionIn 1931, Simpson et al. coined the term “peritoneal carcinomatosis” to describe the regional spread of ovarian tumors as localized or extended with involvement of the peritoneal serous membrane and neighboring anatomical structures.Research into the origin of peritoneal carcinomatosis is based on two phases in a woman's life: Embryo developmentDuring week 3, the bilaminar disc becomes a trilaminar disc called the mesoderm. Inside the lateral plate mesoderm, the coelomic cavity is divided into 2 layers: the parietal (somatic) mesoderm, which gives rise to the parietal peritoneum and pleural surfaces; and the visceral (splanchnic) mesoderm, which gives rise to the visceral peritoneum, visceral surface of the pleura, gonadal stroma, and the muscular layer of the hollow viscera and its mesenteries. Tumor spreadTranscoelomic metastasis and metaplasia of pluripotent stem cells in the peritoneum was involved in the pathogenesis of ovarian cancer. This involvement takes the form of a synchronous malignant transformation at multiple foci and may cause intraperitoneal field cancerization.Pluripotent stem cells play a role both in the development of the embryonic peritoneum and in the spread of transcoelomic tumors. Consequently, knowledge of the origin of these cells (embryonic or current) could be extremely useful.The many markers that act during the embryonic period can affect descendants, that is, cells are already marked before specification and differentiation are activated. Thus, programmed activation could be attributed to genetic and epigenetic changes.

Masking ability of bi- and tri- laminate all-ceramic veneers on tooth-colored ceramic discs.

A predictable esthetic outcome is imperative when placing ceramic veneers. Discolored teeth pose a major challenge as sufficient material thickness is required to achieve a good esthetic result. There is limited evidence in the literature that compares the masking ability of multi-laminate veneers. The aim of this in-vitro study was to compare the masking ability of bi-laminate (BL) and tri-laminate (TL) all-ceramic veneers cemented on tooth-colored ceramic discs. A total of 40 veneers (shade A1, 10-mm diameter, 0.8-mm thick) were manufactured-20 BL veneers (0.4-mm pressable ceramic coping veneered with 0.4-mm thick enamel layer) and 20 TL veneers (0.4-mm coping veneered with 0.2-mm thick opaque interlayer and 0.2-mm thick enamel layer). A bonding apparatus was utilized to adhesively cement all veneers on the ceramic discs (shade A1), simulating teeth of light and dark color. The resulting groups (N = 10 each) were the reference groups (shade A1 ceramic base) BL-1 and TL-1 veneers, and the test groups (shade A4 ceramic base) BL-4 and TL-4 veneers. The color of the cemented veneers was measured using a spectrophotometer. The data were converted to CIE L*a*b* coordinates, and ΔE* were calculated to allow for statistical analysis. The color differences between the samples with the A1 and A4 ceramic bases were significantly lower when covered with TL veneers (mean ΔE*: 3.2 units) than with BL veneers (mean ΔE*: 4.0 units: p < 0.001), indicating a better masking ability of the TL veneers. The 0.8-mm thick TL veneer was able to mask darker tooth-colored ceramic disc within clinically acceptable limits. Increased understanding of the masking ability of ceramics and of color science is necessary in these esthetically aware times. Providing tri-laminate veneers for darker colored teeth seems to result in more predictable esthetical results than when using bi-laminate veneers. Patients with discolored/darker teeth may benefit from a more predictable esthetic result when teeth restored with tri-laminate rather than bi-laminate veneers.

Differentiation of Mouse Embryonic Stem Cells into Endoderm without Embryoid Body Formation

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.

Uterine expression of cp4 gene homolog in the Stripe‐faced Dunnart, Sminthopsis macroura: Relationship with conceptus development and progesterone profile

The marsupial conceptus is surrounded by a uterine-secreted shell coat for 60-80% of gestation. Coat protein 4 (CP4) is the only marsupial shell coat protein characterized and it has only been identified in one species, the Common Brushtail Possum. In this possum, uterine transcription and secretion of cp4 during the oestrous cycle is biphasic and associated with the stage of conceptus development. Here we cloned cp4 (sm-cp4) from a distantly related species, the Stripe-faced Dunnart (Sminthopsis macroura). Transcription of sm-cp4 and secretion of smCP4 were identified by semi-quantitative RT-PCR and immunohistochemistry, respectively. The effect of reproductive hormones on sm-cp4 transcription was investigated in vitro by treatment of uterine explant cultures with oestrogen and/or progesterone. In vivo, uterine expression of smCP4 was biphasic and associated with conceptus development. Uterine smCP4 expression (transcription and secretion) began during the pre-ovulatory period and continued post-ovulation during cleavage stages. Transcription of sm-cp4 continued during the unilaminar blastocyst stage, but smCP4 secretion was reduced during this stage. During the bilaminar blastocyst stage, both transcription of sm-cp4 and secretion of smCP4 were low before they both resumed during the trilaminar blastocyst stage, and continued during the embryo and fetal stages. In vitro uterine transcription of sm-cp4 increased after incubation with progesterone.

Viewpoint 3

Viewpoint 3

Identification and temporospatial distribution of bovine primordial germ cells prior to gonadal sexual differentiation.

The temporospatial distribution of bovine primordial germ cells was studied in 34 embryos (18 to 39 days). For a reliable identification of bovine primordial germ cells in varying localizations and at different developmental stages the alkaline phosphatase reaction combined with the use of selected lectins was applied. The first potential primordial germ cells were identified in an 18-day-old trilaminar embryo in the caudal wall of the proximal yolk sac at a distance of less than 100 microm from the germ disc. These cells are alkaline phosphatase-positive. but do not yet react with lectins. From 18 through 23 days, morphogenetic folding converts the flat trilaminar disc into a cylindrical embryonic body. During this folding process primordial germ cells located in the proximal yolk sac area are incorporated into the embryo when this portion of the yolk sac becomes the hind- and mid-gut. Consequently, in 23- to 25-day-old embryos putative primordial germ cells (alkaline phosphatase- and lectin-positive) are situated predominantly in the axial body region at the level of the mesonephros. When the gonadal ridge develops in this region (about day 27) it contains a certain number of primordial germ cells present from the very beginning. Thus, the assumptions of a long-range chemoattraction of primordial germ cells by the gonadal ridge, of active immigration from an extraembryonic site. or of a passive transportation via the blood stream are not necessary to explain the initial settlement of bovine primordial germ cells in the gonadal ridge. Within the gonadal ridge (days 27-31) and later in the still sexually indifferent gonadal fold (32-39 days) the primordial germ cells are unevenly distributed. Extragonadal potential primordial germ cells (alkaline phosphatase-positive, but with reduced or no lectin staining) are regularly present in large numbers in bovine embryos with indifferent gonads. Such cells occur predominantly in the paraaortal tissue and in the liver, but also in the branchial arches. The different locations of extragonadal primordial germ cells are discussed in the light of recent evidence that germ cells and haematopoietic cells share common ancestors.

Origins of pluriblast and trophoblast in the eutherian conceptus.

Growth, differentiation and morphogenesis during the early development of amniotic vertebrates are concerned primarily with the establishment of a system of extraembryonic membranes. These supporting tissues are limited to higher vertebrates and have no obvious homologues in invertebrates. They surround and support a relatively small and emergent population of pluripotential cells, within which the embryo and from which the fetus will develop. These cells become organized in a trilaminar disc comprising ectoderm, mesoderm and endoderm (Fig. I), which develops from an epiblastic stem cell population at gastrulation (Cruz et al. 1996). Thus, in eutherian mammals, embryonic cells are not present until relatively late, at about 14-18 days after fertilization in the human and 6-7 days in the mouse. This period from fertilization to gastrulation and the appearence of the embryo is therefore described as being embryogenic (for discussion of the terminology used in this paper see Johnson and Selwood 1996).

Endoderm cells of the equine yolk sac from Day 7 until formation of the definitive yolk sac placenta

SummaryOn Day 7 of pregnancy, when the inner cell mass coalesces to one side of the blastocyst, a discontinuous layer of cells is present underlying the inner cell mass and isolated cells were found around the mural trophoblast. As early as Day 8, these cells that were scattered around the blastocyst cavity unite to form a continuous endodermal layer, resulting in a bilaminar blastocyst. The endodermal cells have slightly bulging apical surfaces towards the cavity of the blastocyst, but in many areas have flanges and projections that form complex basolateral compartments within the endoderm and especially between the endoderm and trophoblast. These cells also develop an extensive series of unusual apical ridges. With the development of the primitive streak and the trilaminar blastocyst, angiogenesis proceeds from the forming embryo into the yolk sac. At first the anastomotic vessels form a simple mesh, but by Day 20, erythroblasts are present in the newly formed sinus terminalis. The loss of the capsule (Day 21) and the development of the embryonic circulation establish the relationships of the definitive yolk sac. There is a bilaminar region with endodermal cells showing basolateral compartmentalisation and a specialised sinus terminalis region bordering the choriovitelline placenta. The largest endodermal cells are associated with the choriovitelline placenta, but a substantial cuboidal lining also covers the splanchnopleuric portion of the vascular yolk sac. The allantochorion rapidly assumes predominance in respiratory exchanges, and the choriovitelline placenta not only decreases in relative area but increases in thickness. However, the 3 major regions of the yolk sac persist for several weeks after their formation, and both the vasculature and especially the endodermal cells remain robust despite the decrease in size of the yolk sac. It is concluded that the pleomorphic endodermal cells of the equine yolk sac differentiate in response to local signals. By Day 25 the allantochorion is an increasingly significant exchange unit, and the continued robustness of the vascular portions of the yolk sac and especially its endodermal layer suggests that synthetic and metabolic activities of the endodermal cells will be the dominant function of the yolk sac subsequently.

5 Mechanisms Underlying the Development of Pattern in Marsupial Embryos

Publisher Summary This chapter describes the marsupial embryonic development during pre-implantation stages up to the trilaminar blastocyst stage. Because the developing polarity of the marsupial embryo is initiated during oogenesis, the review also covers oogenesis and fertilization in marsupials. The chapter discusses the idea that the marsupial embryo acquires a polarized state that is initially related to the position of the nucleus and the pattern of distribution of cytoplasmic organelles, especially yolky storage products, in oocytes and zygotes. The particular nature of this polarized state generates a particular and highly specific pattern of cleavage. The chapter also discusses the potential role of positional signals, the order of cell division and maternal determinants operating at a two-dimensional level in specifying cell fate, and the early steps in determination.

Early postimplantation embryolethality in mice following in utero inhibition of adenosine deaminase with 2'-deoxycoformycin.

Adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine (or 2'-deoxyadenosine) to inosine (or 2'-deoxyinosine). Previously, we have shown that ADA activity is subject to strong cell-specific developmental regulation in placental tissues of mice between days 6 and 11 of gestation (Knudsen et al.:Biology of Reproduction 39:937-951, 1988). In the present study, we examined the effects of intrauterine exposure to 2'-deoxycoformycin (dCF; pentostatin), a potent irreversible inhibitor of ADA, on early postimplantation development. Deoxycoformycin was administered to pregnant ICR mice as a single intraperitoneal injection at a dose of 5 mg/kg on one of days 6 through 11 of gestation (plug day 0). A marked increase in the incidence of implantation site resorptions was observed following treatment specifically on days 7 (61% resorbed) or 8 (78% resorbed). No effect was observed following treatment on days 6, 9, 10, or 11. ADA-immunoreactive protein was shown, by ABC-immunoperoxidase staining on days 7 or 8 of gestation, to be present at high levels in decidual cells of the antimesometrial region but at below-detectable levels in the embryo. Treatment of pregnant dams with dCF on day 7 produced a complete (greater than 99%) inhibition of ADA activity in the antimesometrial decidua by 30 min, induced excessive cell death in the prospective neural plate and primary mesenchyme of the trilaminar disc by 6 h, and arrested embryonic development at an early somite stage. These results suggest that the antimesometrial decidua plays a protective role in preventing an inappropriate accumulation of endogenous ADA substrates in the implantation site.

Differentiation of the embryonic disc, amnion, and yolk sac in the rhesus monkey

The inner cell mass of the blastocyst has differentiated into epiblast and hypoblast (primitive endoderm) prior to implantation. Since endoderm cells extend beyond the epiblast, it can be considered that both parietal and visceral endoderm are present. At implantation, epiblast cells begin to show marked evidence of polarity. They form a spherical aggregate with their basal ends toward the basal lamina and apical ends toward the interior. The potential for an internal space is formed by this change in polarity of the cells. No cytological evidence of separation of those cells that will form amniotic epithelium from the rest of the epiblast is seen until a cavity begins to form. The amniotic epithelium is originally contiguous with overlying cytotrophoblast, and a diverticulum remains in this position during early development. Epiblast forms a pseudostratified columnar epithelium, but dividing cells are situated toward the amniotic cavity rather than basally. The first evidence of a trilaminar disc occurs when a strand of cells contiguous with epiblast is found extending toward visceral endoderm. These presumptive mesoderm cells are undifferentiated, whereas extraembryonic mesoderm cells are already a distinct population forming extracellular materials. After implantation, visceral endoderm cells proliferate forming an irregular layer one to three cells thick. Visceral endoderm cells have smooth apical surfaces, but very irregular basal surfaces, and no basal lamina. At the margins of the disc, visceral endoderm is continuous with parietal endoderm and reflects back over the apices of the marginal visceral endoderm cells. This sacculation by visceral endoderm cells precedes pinching off of the secondary yolk sac from the remaining primary yolk sac.

Prenatal Development after Diapause in the Marsupial Macropus rufogriseus

Prenatal development of Macropus rufogriseus was described in eight stages ranging from a quiescent blastocyst to a near-term foetus. Embryos and corpora lutea were induced to resume development after diapause by the removal of suckling pouch young (RPY). The diameter of unilaminar blastocysts ranged from approximately 260 �m at 0 days after RPY to 290 �m after 6 days. At 9 days after RPY (330 �m diameter) the zona pellucida and mucoid coat were apparently absent and endoderm had proliferated (early bilaminar). At 15 days, mesoderm had formed in the trilaminar blastocyst (4.8 mm diameter). By 18 days after RPY the shell membrane had ruptured and was 3.0 �m in thickness (7.0 pm at 0 days after RPY). At this time the pre-foetal embryo had acquired forelimb ridges, mesonephric kidneys, optic stalks and 25 paired somites; persistent features included an amniopore, posterior neuropore and an intact stomodeal membrane; the lung buds and allantois were rudimentary; cervical, lumbosacral and cranial flexures were present. By 20 days after RPY, digital anlagen were noted in the forelimb paddles of the pre-foetal embryo (32 paired somites); the gonadal ridges; glomeruli and optic cups had formed. At 25 days after RPY, the near-term foetus had forelimbs with claws on the separate digits, hindlimb paddles, pontine flexure and cardiac valves; the metanephric kidneys, pigmented and nervous retinas and villous intestine were present. A single birth was recorded at 26 days after RPY (delayed gestation length).

Observations on twin baboon embryos (Papio sp.).

AbstractTwin baboon embryos were acquired by hysterotomy from a multipara on the twenty‐sixth day of gestation. All preceding and succeeding pregnancies yielded single births. This case of dichorionic, diamniotic twins is unique because there is a seven day difference in the morphological development of the two embryos. The embryos are approximately the same fertilization age because mating was limited to one 12 hour period of the menstrual cycle. In addition only one corpus luteum was identified at laparotomy. Both twins were males as indicated by the sex chromatin, eliminating the positive identification of dizygosity. Several alternatives for the twins' origin are cited. One possibility is that there was a division of the blastomeres at the two‐cell stage, or at some other stage before implantation, yielding monozygotic twins. Another possibility is that both ova arose from the same follicle, yielding dizygotic twins. The larger embryo is 2.0 mm in greatest length and has six pairs of somites, and is similar to ten other baboon embryos 25–26 days old. The smaller embryo is of the trilaminar disc stage and is comparable in developmental features to five other baboon embryos 18–19 days old. The morphological features of each placenta are comparable in development to the age of its respective embryo.