Embryogenesis Differentiation Research Articles

PATH-57. WHOLE EXOME SEQUENCING OF INTRACRANIAL EPIDERMOID CYSTS (IECS) REVEALS POTENTIAL SOMATIC DRIVERS

Abstract Intracranial Epidermoid Cysts (IECs) are benign, slow-growing, asymptomatic tumors with an incidence rate of 1% of all intracranial tumors. They are significantly enlarged when discovered and can cause seizures. IECs are suspected to form due to disruptions in neural tube closure. Given their rarity, formation of IECs is under-investigated and surgery is the only treatment option. However, because the cyst lining can be adherent to important brain structures, resection is challenging and can result in post-operative complications, highlighting the need to investigate the pathogenesis of IECs to develop drug therapeutic options. We performed paired-end Whole Exome Sequencing (WES) on DNA from six sporadic IECs. Data processing of raw unmapped reads was performed in tumor-normal and tumor-only mode as appropriate, following GATK best practices for variant calling. We identified 2,850 SNPs and 2,102 indels in this cohort. We identified two common variants, NKX2.5 (p.P214del) and STIM-1 (p.T576del, p.T502del) across all samples, which is a novel finding. We also identified 27 genes and variants shared variably across the samples. Gene enrichment analysis revealed that the Sonic Hedgehog (SHH) pathway is implicated in these gene sets due to activating mutations in the SMO gene (p.L23del). We are using an immunohistochemical stain to validate this finding. The mutant NKX2.5, a transcription factor (TF) responsible for stem-cell differentiation in embryogenesis, strongly indicates that the tumor microenvironment (TME) is disrupted. Deletions in the STIM-1 gene indicate loss of interaction with Orai1 calcium channels, disrupting store operated Ca2+ entry (SOCE) and preventing typical cell migration, resulting in trapped fibroblasts. Digital droplet PCR (ddPCR) of these samples is underway to validate the low tumor allele frequencies (< 4%). The discovery of activating mutations in SMO is significant because it potentiates FDA-approved SMO-inhibitors like Vismodegib and Sonidegib as viable treatment options for patients with IECs.

ASB2 is a novel E3 ligase of SMAD9 required for cardiogenesis

Cardiogenesis requires the orchestrated spatiotemporal tuning of BMP signalling upon the balance between induction and counter-acting suppression of the differentiation of the cardiac tissue. SMADs are key intracellular transducers and the selective degradation of SMADs by the ubiquitin–proteasome system is pivotal in the spatiotemporal tuning of BMP signalling. However, among three SMADs for BMP signalling, SMAD1/5/9, only the specific E3 ligase of SMAD9 remains poorly investigated. Here, we report for the first time that SMAD9, but not the other SMADs, is ubiquitylated by the E3 ligase ASB2 and targeted for proteasomal degradation. ASB2, as well as Smad9, is conserved among vertebrates. ASB2 expression was specific to the cardiac region from the very early stage of cardiac differentiation in embryogenesis of mouse. Knockdown of Asb2 in zebrafish resulted in a thinned ventricular wall and dilated ventricle, which were rescued by simultaneous knockdown of Smad9. Abundant Smad9 protein leads to dysregulated cardiac differentiation through a mechanism involving Tbx2, and the BMP signal conducted by Smad9 was downregulated under quantitative suppression of Smad9 by Asb2. Our findings demonstrate that ASB2 is the E3 ligase of SMAD9 and plays a pivotal role in cardiogenesis through regulating BMP signalling.

Evolutionary-histological studies of the doctor of medicine Vera Mikhailovna Danchakova

The historical article presents material that analyzes the contributor from the first female histologists V.M. Danchakova in the study of problems of evolutionary histology and questions of normal and pathological histogenesis. Vera Mikhailovna Danchakova is a student of Professor A.A. Maksimov, the first woman, who defended a histological dissertation at the Imperial Military Medical Academy (1907). In foreign literature, her work is referred to as pioneering in the study of cells, which are now called stem cells. V.M. Danchakova made a significant contribution to the study of the evolutionary aspects of histogenesis of tissues with various cambial properties, for the first time showed the extragonadal origin of primary germ cells using the example of birds, and obtained new additional experimental data confirming A.A. Maksimov's unitary theory of hematopoiesis. Thanks to her research, the possibility of malignancy of stem and progenitor cells was shown for the first time. V.M. Danchakova was prompted by the study of generative cells (primary germ cells, gonocytes), as well as the influence of male and female sex hormones on sex differentiation in embryo-genesis. The results of her experiments using the method of tissue and cell cultures formed the basis for the development of the doctrine of the immune response "graft versus host”.

Morphological characteristics of mucinous adenocarcinoma of the colon and its embryogenetic premises

This review reveals the anatomical, physiological and embryogenetic features of the proximal colon to explain the reasons for such frequent localization of mucinous phenotype of adenocarcinoma (MAC) here. It was shown that later differentiation in embryogenesis causes relative insufficiency of the proximal part as a structure of the digestive and immune systems. Physiologically lower lymphoid tissue (GALT) density here leads to the formation of a certain composition of the intestinal microbiota, which is different from that in the distal part, which is a significant link in the etiopathogenesis of proximal colon cancer. It is confirmed also by different composition of biofilms on the surface of epithelial tumors in the right and left halves of the colon, as well as differences in the molecular mechanisms of carcinogenesis, depending on the location of the cancer. Common for proximal part genetic changes, called as CIMP-phenotype, microsatellite instability and BRAF proto-oncogene mutation lead to excessive secretion of specific mucin fractions (mainly MUC2 and MUC5AC), the imbalance of its composition and the formation of MAC. An earlier age of onset, frequent association with hereditary non-polypous colorectal carcinoma, the predominance of MUC2 and MUC5AC fractions, similar to the embryonic period, as well as a higher level of cancer-embryonic antigen in patients with MAC indicate the influence of anatomical, physiological and embryogenetic features of the proximal colon on carcinogenesis long before its formation. Thus, a detailed understanding of MAC carcinogenesis is necessary for an adequate assessment of its effective prevention in time, as well as dealing with it as with specific nosological unit requiring specific treatment principles.

Apical cytoskeletons and junctional complexes as a combined system in epithelial cell sheets.

Epithelial cell sheet formation is central to many aspects of vertebrate development and function. For example, it is a major principle of differentiation in embryogenesis and regeneration, enables the compartmentalization of tissues, and is the basis for the maintenance of homeostasis throughout the body. A key characteristic of biologically functional epithelial cell sheets is a clear difference between the top and bottom sides owing to the apicobasal polarity of the cells in the sheet, as seen in the simple polar epithelia. Epithelial cell sheets are formed by cell-cell adhesion conferred by junctional complexes, in particular via tight junctions (TJs), which thus create a paracellular barrier. This review focuses on the apical side of the sheet, which serves as the front line. The apical membranes and TJs of the various tissues have specific characteristics that enable them to function and adapt to their biological context: each system must be robust, but also dynamic and flexible to maintain homeostasis. Here, we describe various apical cytoskeletal structures that are critical to the integrity of epithelial cell sheets. We also discuss the association of apical cytoskeletal networks with TJs, which thus forms a combined system, tentatively termed the TJ-apical complex.

The effect of ivabradine and ω-3 polyunsaturated fatty acids on the fibronectin plasma levels in patients with heart failure

Chronic heart failure (HF) is a highly prevalent chronic pathological condition with high morbidity and mortality rates. The cost for HF treatment is 2% of the national health expenditures each year. Fibronectin (Fn) has a crucial role in the process of tissue-specific morphogenesis and cell differentiation in embryogenesis; it stimulates fibroblasts and different growth factors during wound healing and tissue reparation. The aim of our study was to assess the possible effects of Ivabradine and ω-3 polyunsaturated fatty acids on the fibronectin plasma level in patients with heart failure. 357 Patients with ischemic HF and the sinus rhythm were observed. In accordance to treatment regimens all patients were divided into four groups: group I – basic treatment; group II – basic treatment and Ivabradine; group III – basic treatment and PUFA; group IV – basic treatment with Ivabradine and PUFA. The fibronectin levels were measured by ELISA method. The average value of the Fn concentration in HF patients was 1.25 times higher than in the control group (p<0.05). In patients with basic HF treatment the tendency to the Fn plasma level decrease (p>0.05) was observed. In the group with additional prescription of Ivabradine the concentration of this protein in the blood decreased by 14.5% (p<0.01). The same situation was observed in the group with additional use of PUFA – by 11.1% (p<0.05). In group IV the plasma concentration of Fn decreased by 12.9% (p<0.05). Thus, Ivabradine and ω-3 polyunsaturated fatty acids, either alone or in combination, decrease the plasma levels of fibronectin in patients with heart failure, and it shows a pronounced cardioprotective effect.

Evolutionary roots of arginase expression and regulation.

Two main types of macrophage functions are known: classical (M1), producing nitric oxide, NO, and M2, in which arginase activity is primarily expressed. Ornithine, the product of arginase, is a substrate for synthesis of polyamines and collagen, important for growth and ontogeny of animals. M2 macrophages, expressing high level of mitochondrial arginase, have been implicated in promoting cell division and deposition of collagen during ontogeny and wound repair. Arginase expression is the default mode of tissue macrophages, but can also be amplified by signals, such as IL-4/13 or transforming growth factor-β (TGF-β) that accelerates wound healing and tissue repair. In worms, the induction of collagen gene is coupled with induction of immune response genes, both depending on the same TGF-β-like pathway. This suggests that the main function of M2 “heal” type macrophages is originally connected with the TGF-β superfamily of proteins, which are involved in regulation of tissue and organ differentiation in embryogenesis. Excretory–secretory products of metazoan parasites are able to induce M2-type of macrophage responses promoting wound healing without participation of Th2 cytokines IL-4/IL-13. The expression of arginase in lower animals can be induced by the presence of parasite antigens and TGF-β signals leading to collagen synthesis. This also means that the main proteins, which, in primitive metazoans, are involved in regulation of tissue and organ differentiation in embryogenesis are produced by innate immunity. The signaling function of NO is known already from the sponge stage of animal evolution. The cytotoxic role of NO molecule appeared later, as documented in immunity of marine mollusks and some insects. This implies that the M2-wound healing promoting function predates the defensive role of NO, a characteristic of M1 macrophages. Understanding when and how the M1 and M2 activities came to be in animals is useful for understanding how macrophage immunity, and immune responses operate.

Wnt-Catenin Signaling System Functions in Embryoid Bodies Aggregated from Human Embryonic Stem Cell

As an essential molecule in Wnt/β-catenin signaling, β-Catenin plays a crucial role in the decision making for tissue differentiation in embryogenesis and pathogenesis. The signaling was implicated in the development of skin. In mouse skin, the deferential fate of the skin stem cell depends on β-catenin, which organizes stem cells into follicular or epidermal lineages. These analyses indicate that Wnt/β-catenin signaling should also function in the development and differentiation of human embryonic stem cell. To study the function of β-catenin in early differentiation of human stem cells, we cultured H9 stem cell and aggregated them into embryoid bodies (EB). In this study, we revealed that in early EBs some guarding cells were first differentiated from EB stem cell aggregates. These early differentiated cells for guarding epithelial cells have strongest expression of β-catenin within EB. These cells were flattened on the surface of EB, covering the surface by connections formed through protein interactions. At certain confocal sections of EB, instead of a round boundary, a polygonal boundary was observed even though the EB appeared round under conventional microscope. In these polygonal boundaries, β-catenin positive guarding epithelial cells were positioned on every corner of the polygon. In the inner portion of the EB, undifferentiated β-catenin positive cells express β-catenin in the nucleus. As the initially simple shape of EB becomes more and more intricate during development, we revealed that more β-catenin positive cells were also observed in this complex structure. Based on these results, we predicted β-catenin to play different roles while guarding epithelial cells or undifferentiated stem cells in the inner portion of EB in in vitro culture system. Phosphorylation of β-catenin may be a critical factor for fate determination of the human stem cell.

Neuronal cell death during metamorphosis of Hydractina echinata (Cnidaria, Hydrozoa)

In planula larvae of the invertebrate Hydractinia echinata (Cnidaria, Hydrozoa), peptides of the GLWamide and the RFamide families are expressed in distinct subpopulations of neurons, distributed in a typical spatial pattern through the larval body. However, in the adult polyp GLWamide or RFamide-expressing cells are located at body parts that do not correspond to the prior larval regions. Since we had shown previously that during metamorphosis a large number of cells are removed by programmed cell death (PCD), we aimed to analyze whether cells of the neuropeptide-expressing larval nerve net are among those sacrificed. By immunohistochemical staining and in situ hybridization, we labeled GLWamide- and RFamide-expressing cells. Double staining of neuropeptides and degraded DNA (TUNEL analysis) identified some neurosensory cells as being apoptotic. Derangement of the cytoplasm and rapid destruction of neuropeptide precursor RNA indicated complete death of these particular sensory cells in the course of metamorphosis. Additionally, a small group of RFamide-positive sensory cells in the developing mouth region of the primary polyp could be shown to emerge by proliferation. Our results support the idea that during metamorphosis, specific parts of the larval neuronal network are subject to neurodegeneration and therefore not used for construction of the adult nerve net. Most neuronal cells of the primary polyp arise by de novo differentiation of stem cells commited to neural differentiation in embryogenesis. At least some nerve cells derive from proliferation of progenitor cells. Clarification of how the nerve net of these basal eumetazoans degenerates may add information to the understanding of neurodegeneration by apoptosis as a whole in the animal kingdom.

Physical plasticity of the nucleus in stem cell differentiation

Cell differentiation in embryogenesis involves extensive changes in gene expression structural reorganization within the nucleus, including chromatin condensation and nucleoprotein immobilization. We hypothesized that nuclei in naive stem cells would therefore prove to be physically plastic and also more pliable than nuclei in differentiated cells. Micromanipulation methods indeed show that nuclei in human embryonic stem cells are highly deformable and stiffen 6-fold through terminal differentiation, and that nuclei in human adult stem cells possess an intermediate stiffness and deform irreversibly. Because the nucleo-skeletal component Lamin A/C is not expressed in either type of stem cell, we knocked down Lamin A/C in human epithelial cells and measured a deformability similar to that of adult hematopoietic stem cells. Rheologically, lamin-deficient states prove to be the most fluid-like, especially within the first approximately 10 sec of deformation. Nuclear distortions that persist longer than this are irreversible, and fluorescence-imaged microdeformation with photobleaching confirms that chromatin indeed flows, distends, and reorganizes while the lamina stretches. The rheological character of the nucleus is thus set largely by nucleoplasm/chromatin, whereas the extent of deformation is modulated by the lamina.

Transglutaminase 3 expression in C57BL/6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornified envelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR. TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then we determined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. We found a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cells from E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed in the granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin development provided several morphological evidences for the epidermal differentiation. The above findings suggest that the expression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.

Estradiol‐17β content in developing eggs and induced sex reversal of the medaka (Oryzias latipes)

To clarify the effect of exogenous estradiol-17beta (E2) on sex differentiation, the E2 content of developing eggs of Oryzias latipes was measured by radioimmunoassay. Endogenous E2 was present in lower concentrations in ovulated, mature eggs in the ovarian cavity than in intrafollicular oocytes. The E2 content of eggs precipitously declined to a minimum level by 2 days post-fertilization. The E2 content of eggs was affected by 24 hr of incubation in medium containing exogenous E2 at concentrations above 10 ng/ml. Short (24 hr) exposure of fertilized eggs in the early developmental stage to exogenous E2 at concentrations of 10 ng/ml induced sex reversal of some genotypic males to functional females. However, endogenous E2 levels in fertilized eggs might not influence sexual differentiation in embryogenesis. The present results suggest the possibility that concentrations of exogenous E2 higher than that of endogenous E2 triggers a priming step in the cascade of sex differentiation toward the female, and this effect is maintained.

Effects of Radiation on the Developing Rice Embryos. I. Cytohistological and Histological Observations of lrradiated Embryos.

The rice plants at the different developmental stages of embryo were exposed to X-rays, the dose being 3, 6, 12 and 20 kR at any of the stages. The main results obtained were as follows : 1) The growth retardation in length and width of embryo by irradiation varied considerably with the development stage of embryo at the time of irradiation, the younger the embryo, the effects being the more severe. 2) The common cellular abnormalities observed after irradiation were the inhibition of nuclear division, extraordinary enlargement and vacuolation of cells, thickening of cell wall and degeneration of embryo. In general, these effects increased in degree and frequency with increasing radiation doses. 3) Especially when given irradiation dose was the higher dose, the observed vegetative prolifera-tiori accompanied by disruption of organized embryo were the more often. 4) The histological and morphological abnorma-lities produced by irradiation varied considerably depending on the stage of development of embryo at the time of irradiation, the most effective stage being the stage of the onset of differentiation in embryogenesis. 5) No definite conclusions as to the recovery process following irradiation could be drawn from the results of the experiments. However, it was supposed that in the case where irtadiations were carried out with the lower doses and at the later stages of embryogenesis, the cellular recovery seemed to occur the more predominantly. On the other hands, the replacement by division of the adjacent cells seemed to occulr the more predomina-ntly in the case where irradiation was applied with the higher dose and at the earlier stages of emb-ryogenesis.