Yolk Cell Research Articles

Male reproductive system of the deep-sea acorn worm Quatuoralisia malakhovi (Hemichordata, Enteropneusta, Torquaratoridae) from the Bering Sea

BackgroundThe deep-sea acorn worm Quatuoralisia malakhovi belongs to the phylum Hemichordata, class Enteropneusta, family Torquaratoridae, which was described in 2005. Owing to their epibenthic lifestyle and deep-sea habitat features, torquaratorids differ anatomically from shallow-water acorn worms; however, their morphology and fine structure are poorly studied. We have the opportunity to make three complete detailed series of histological sections of Q. malakhovi and to study the microscopic anatomy, histology and fine structure of the reproductive system of this acorn worm using scanning and transmission electron microscopy.ResultsThe sexes of Q. malakhovi are separate and indistinguishable externally. The lobed testes occupy the dorsal side of the genital wings and distinctly bulge into the peribranchial cavity by their mature lobes. The central part of the testis is always submerged into the genital wing and opens via a single gonad pore. The monociliary muscle cells stretch along the external wall of the testis and surround the gonad pore, probably taking part in the contraction of the testis lobes for spawning. The germinative epithelium of the testis contains spermatogenic cells at different stages of development and interstitial cells. Yolk cells are not found. Interstitial cells embrace the spermatogonia and spermatogenic columns, providing horizontal compartmentalization of the germinative epithelium, and contain numerous phagosomes with remnants of degenerating spermatogenic cells. The testis wall contains haemal lacunae, which are usually located on the side opposite the gonad pore. We describe the fine structure of spermatogonia, spermatocytes clustered in spermatogenic columns, spermatids, and spermatozoa. Spermatozoa are of the ectaquasperm type and consist of an acorn-shaped head and a flagellum 18–25 µm long. The sperm head includes a beak-shaped acrosomal part, a spherical nucleus and a midpiece containing a ring of 5 or rarely 6 mitochondria.ConclusionsThe male reproductive system and sperm structure of Q. malakhovi, a representative of the family Torquaratoridae, have a number of differences from shallow-water acorn worms; however, the spermatogenesis and sperm structure of Q. malakhovi generally follow the pattern of the other three enteropneust families, and the phylogenetic significance of these deviations should be the subject of further research.

Zebrafish ApoB-Containing Lipoprotein Metabolism: A Closer Look.

Zebrafish have become a powerful model of mammalian lipoprotein metabolism and lipid cell biology. Most key proteins involved in lipid metabolism, including cholesteryl ester transfer protein, are conserved in zebrafish. Consequently, zebrafish exhibit a human-like lipoprotein profile. Zebrafish with mutations in genes linked to human metabolic diseases often mimic the human phenotype. Zebrafish larvae develop rapidly and externally around the maternally deposited yolk. Recent work revealed that any disturbance of lipoprotein formation leads to the accumulation of cytoplasmic lipid droplets and an opaque yolk, providing a visible phenotype to investigate disturbances of the lipoprotein pathway, already leading to discoveries in MTTP (microsomal triglyceride transfer protein) and ApoB (apolipoprotein B). By 5 days of development, the digestive system is functional, making it possible to study fluorescently labeled lipid uptake in the transparent larvae. These and other approaches enabled the first in vivo description of the STAB (stabilin) receptors, showing lipoprotein uptake in endothelial cells. Various zebrafish models have been developed to mimic human diseases by mutating genes known to influence lipoproteins (eg, ldlra, apoC2). This review aims to discuss the most recent research in the zebrafish ApoB-containing lipoprotein and lipid metabolism field. We also summarize new insights into lipid processing within the yolk cell and how changes in lipid flux alter yolk opacity. This curious new finding, coupled with the development of several techniques, can be deployed to identify new players in lipoprotein research directly relevant to human disease.

Sturgeon gut development: a unique yolk utilization strategy among vertebrates.

In vertebrates, maternally supplied yolk is typically used in one of two ways: either intracellularly by endodermal cells or extracellularly via the yolk sac. This study delves into the distinctive gut development in sturgeons, which are among the most ancient extant fish groups, contrasting it with that of other vertebrates. Our observations indicate that while sturgeon endodermal cells form the archenteron (i.e., the primitive gut) dorsally, the floor of the archenteron is uniquely composed of extraembryonic yolk cells (YCs). As development progresses, during neurulation, the archenteric cavity inflates, expands laterally, and roofs a semicircle of YCs. By the pharyngula stage, the cavity fully encompasses the YC mass, which begins to be digested at the hatching stage. This suggests a notable deviation in sturgeon gut development from that in other vertebrates, as their digestive tract initiates its function by processing endogenous nutrition even before external feeding begins. Our findings highlight the evolutionary diversity of gut development strategies among vertebrates and provide new insights into the developmental biology of sturgeons.

A rare case of yolk cell carcinoma presenting as ovarian torsion

A rare case of yolk cell carcinoma presenting as ovarian torsion

Drosophila embryos allocate lipid droplets to specific lineages to ensure punctual development and redox homeostasis.

Lipid droplets (LDs) are ubiquitous organelles that facilitate neutral lipid storage in cells, including energy-dense triglycerides. They are found in all investigated metazoan embryos where they are thought to provide energy for development. Intriguingly, early embryos of diverse metazoan species asymmetrically allocate LDs amongst cellular lineages, a process which can involve massive intracellular redistribution of LDs. However, the biological reason for asymmetric lineage allocation is unknown. To address this issue, we utilize the Drosophila embryo where the cytoskeletal mechanisms that drive allocation are well characterized. We disrupt allocation by two different means: Loss of the LD protein Jabba results in LDs adhering inappropriately to glycogen granules; loss of Klar alters the activities of the microtubule motors that move LDs. Both mutants cause the same dramatic change in LD tissue inheritance, shifting allocation of the majority of LDs to the yolk cell instead of the incipient epithelium. Embryos with such mislocalized LDs do not fully consume their LDs and are delayed in hatching. Through use of a dPLIN2 mutant, which appropriately localizes a smaller pool of LDs, we find that failed LD transport and a smaller LD pool affect embryogenesis in a similar manner. Embryos of all three mutants display overlapping changes in their transcriptome and proteome, suggesting that lipid deprivation results in a shared embryonic response and a widespread change in metabolism. Excitingly, we find abundant changes related to redox homeostasis, with many proteins related to glutathione metabolism upregulated. LD deprived embryos have an increase in peroxidized lipids and rely on increased utilization of glutathione-related proteins for survival. Thus, embryos are apparently able to mount a beneficial response upon lipid stress, rewiring their metabolism to survive. In summary, we demonstrate that early embryos allocate LDs into specific lineages for subsequent optimal utilization, thus protecting against oxidative stress and ensuring punctual development.

Embryonic Development of Parthenogenetic and Sexual Eggs in Lower Termites.

Worldwide, termites are one of few social insects. In this research, the stages of embryonic development in the parthenogenetic and sexual eggs of Reticulitermes aculabialis and R. flaviceps were observed and described. In R. flaviceps, the egg development of the FF and FM groups happened during the early phases of development, whereas in R. aculabialis, this appeared mainly during the late phase of development. The variance in the number of micropyles between the R. flaviceps FF colony type and the R. aculabialis FF colony type was statistically significant. Five stages of egg development were found in both types of R. aculabialis but only the sexual eggs of R. flaviceps. In R. flaviceps, 86% of the parthenogenetic eggs stopped growing during the blastoderm development, with the yolk cell assembling frequently in the center of the egg. According to the results of the single-cell transcriptome sequencing, we investigated the egg-to-larval expression level of genes (pka, map2k1, mapk1/3, hgk, mkp, and pax6) and indicated that the levels of essential gene expression in RaFF were considerably higher than in RfFF (p < 0.05). We also discovered that the oocyte cleavage rate in the FF colony type was considerably lower in R. flaviceps compared to R. aculabialis, which gave rise to a smaller number of mature oocytes in R. flaviceps. During ovulation in both species, oocytes underwent activation and one or two cleavage events, but the development of unfertilized eggs ceased in R. flaviceps. It was shown that termite oocyte and embryonic development were heavily influenced by genes with significant expressions. Results from the databases KEGG, COG, and GO unigenes revealed the control of numerous biological processes. This study is the first to complete a database of parthenogenetic and sexual eggs of R. flaviceps and R. aculabialis.

Features of the microscopic structure of the intestinal wall in females of the ixodic tick of the genus Dermacentor during the feeding period

Ixodic ticks of the genus Dermacentor are parasitomorphic ticks that are carriers of a number of dangerous zooanthropozonoses, have a wide distribution. The intestine of an adult contains several types of cells: digesting, secretory, reserve and yolk cells. Digestive cells of types 1 and 2 are involved in the process of intracellular digestion and are the main components of the wall of the middle intestine. After insemination, yolk cells begin to form in the intestine, regulating the development of oocytes. The research was carried out on the basis of the Department of Parasitology and Veterinary and Sanitary Examination, Anatomy and Pathanatomy named after Prof. S. N. Nikolsky and at the Scientific Diagnostic and Therapeutic Veterinary Center of the Stavropol State Agrarian University, as well as on the basis of veterinary clinics of IP Zaichenko "Veterinary Center named after Pirogova". The object of the study was ixode mites, the material for the study was the digestive system of females, in particular the middle part of the intestine. Macroscopic examination of the nourished females of the Dermacentor tick helped to reveal that the intestine is an unpaired tubular organ divided into three sections: the esophagus, the sac-like meatus and the small intestine. With the arrival of blood, the intestine expands, as a result of which reserve cells begin to grow and differentiate into primary digestive ones. By the time egg laying begins, digestive cells of type I degenerate and are replaced by digestive cells of type II, differentiated from reserve cells. In nourished females, with an active process of vitellogenesis, the presence of specialized yolk cells is noted. Thus, with the beginning of the feeding period in ixodic mites, an increase in the intestine occurs due to the asynchronous development of digestive cells of type I and II. During digestion, secretory cells are focally exfoliated into the intestinal cavity filled with the host's blood, which characterizes the acceleration of the process of erythrocyte hemolysis. In the final phase of digestion, and the beginning of the vitellogenesis process, yolk cells are formed that regulate the development of oocytes.

A hydraulic feedback loop between mesendoderm cell migration and interstitial fluid relocalization promotes embryonic axis formation in zebrafish.

Interstitial fluid (IF) accumulation between embryonic cells is thought to be important for embryo patterning and morphogenesis. Here, we identify a positive mechanical feedback loop between cell migration and IF relocalization and find that it promotes embryonic axis formation during zebrafish gastrulation. We show that anterior axial mesendoderm (prechordal plate [ppl]) cells, moving in between the yolk cell and deep cell tissue to extend the embryonic axis, compress the overlying deep cell layer, thereby causing IF to flow from the deep cell layer to the boundary between the yolk cell and the deep cell layer, directly ahead of the advancing ppl. This IF relocalization, in turn, facilitates ppl cell protrusion formation and migration by opening up the space into which the ppl moves and, thereby, the ability of the ppl to trigger IF relocalization by pushing against the overlying deep cell layer. Thus, embryonic axis formation relies on a hydraulic feedback loop between cell migration and IF relocalization.

Drosophila embryos spatially sort their nutrient stores to facilitate their utilization.

Animal embryos are provided by their mothers with a diverse nutrient supply that is crucial for development. In Drosophila, the three most abundant nutrients (triglycerides, proteins and glycogen) are sequestered in distinct storage structures: lipid droplets (LDs), yolk vesicles (YVs) and glycogen granules (GGs). Using transmission electron microscopy as well as live and fixed sample fluorescence imaging, we find that all three storage structures are dispersed throughout the egg but are then spatially allocated to distinct tissues by gastrulation: LDs largely to the peripheral epithelium, YVs and GGs to the central yolk cell. To confound the embryo's ability to sort its nutrients, we employ Jabba and mauve mutants to generate LD-GG and LD-YV compound structures. In these mutants, LDs are mis-sorted to the yolk cell and their turnover is delayed. Our observations demonstrate dramatic spatial nutrient sorting in early embryos and provide the first evidence for its functional importance.

Marine pollution effects on the reproduction process of Perinereis cultrifera (Annelida, Polychaeta) in Algeria

The Mediterranean Sea is one of the busiest areas worldwide in terms of maritime activity and faces considerable anthropogenic disturbances, such as pollution by hydrocarbons and heavy metals. This study evaluated the environmental status of three sites on the Algerian coast using the annelid polychaete Perinereis cultrifera as a biomonitoring sentinel species. It included different reproductive parameters such as oocyte diameter, frequency distributions, sexual maturity index, biochemical quantitative parameters represented by the vitellogenins and vitellins, and a histological study of the coelome during the reproductive period. The results showed that the females collected from El-Kala (healthy site) have the highest diameter 304.1 ± 24.3 μm during April but the values were increased in Annaba 290.0 ± 21.0 μm and Skikda 245.3 ± 26.7 μm. Significant differences were observed between worms from the three study sites during the three months of the study. Moreover, the size-frequency indicated that a relatively high proportion of females containing mature oocytes was found in April in females collected at El-Kala 52.4%, 46.4% in females collected at Annaba and 36.7% in females collected at Skikda. In addition, regarding the sexual maturity index, the highest values were recorded in April (3.8, 2.9, and 1.9) at the El-Kala, Annaba, and Skikda study sites. However, the biochemical analysis demontrated that the highest mean concentration of vitellogenin was in females of El-Kala (5.3 ± 0.3 µg/mg of coelomic fluid) in April, and the lowest mean concentration was measured in females of Skikda (1.5 ± 0.2 µg/mg of coelomic fluid). Therefore, the highest mean concentration of vitellin was measured in females of El-Kala (4.4 ± 0.2 µg/mg of oocytes) in April, while the lowest mean concentration of vitellin was measured in females of Skikda (1.02 ± 0.2 µg/mg of oocytes) in May. Significant differences were observed between worms from the three sites during the three months of the study. Furthermore, histological observations revealed that oogenesis was asynchronous, with oocytes at different stages of vitellogenesis simultaneously present. The structure of the oocytes of females collected at El-Kala showed intense vitellogenic activity due to the presence of very dense yolk cells. In contrast, the oocytes of the females collected from the two polluted sites (Annaba and Skikda) were small, vitellogenesis presented low intensity, and yolk cells were less frequent at the periphery and less uniform at the cytoplasmic mass; the nucleus was smaller, indicating a slower vitellogenic activity. These observations confirm the previously obtained results.

Revisiting the formation of midgut epithelium in Zygentoma (Insecta) from a developmental study of the firebrat Thermobia domestica (Packard, 1873) (Lepismatidae)

Zygentoma is an order of wingless insects, representing the sister group of Pterygota and constituting Dicondylia together with Pterygota. Contrasting views exist regarding midgut epithelium formation in Zygentoma. According to some reports, in Zygentoma, the midgut epithelium is entirely derived from yolk cells as in other wingless orders; however, according to other reports, the midgut epithelium is of dual origin in Zygentoma, similar to that in Palaeoptera of Pterygota, i.e., the anterior and posterior midgut epithelia are stomodaeal and proctodaeal respectively, whereas the middle part of the midgut originates from yolk cells. Aiming to provide a sound basis to evaluate the true image of midgut epithelium formation in Zygentoma, we examined the formation of the midgut epithelium in detail in Thermobia domestica, and concluded that the midgut epithelium is exclusively derived from yolk cells in Zygentoma, without the stomodaeal and proctodaeal elements involved in its formation. The participation of the anlagen differentiated at or around the stomodaeal and proctodaeal extremities in the formation of the midgut epithelium (bipolar formation) may be regarded as having first appeared not in Dicondylia but in Pterygota, of which the major part is represented by Neoptera with the midgut epithelium formed through bipolar formation.

Asynchronized cell division in embryo-like fossils from the Ediacaran Zhenba microfossil assemblage.

Ediacaran embryo-like spherical fossils exhibit diverse cell adhesion patterns resembling partial cleavage-stage embryos of living animals. Two three-celled specimens characterized by a pair of small cells overlying a large cell have been recovered from the Ediacaran Zhenba microfossil assemblage. Their cell adhesion pattern is highly comparable to a phenomenon reported from the Weng'an biota that was interpreted as fossil embryos undergoing discoidal cleavage. However, our specimens contain fewer cells and thus probably represent developmental precursors of the Weng'an counterparts. Additionally, new material shows several anatomical features that are inconsistent with an embryo interpretation, including (1) an unusually large volume of "blastomeres,"(2) a putative nucleus preserved within the large "yolk cell,"and (3) completely separated cells. Collectively, the Zhenba embryo-like specimens permit a reconstruction of the consecutive developmental sequence from single-celled individuals to the three-celled individuals, leading us to interpret the newly found specimens as products of abnormal development of Ediacaran embryo-like organisms whose affinity remains unresolved.

Determining the role of advection in patterning by bone morphogenetic proteins through neural network model-based acceleration of a 3D finite element model of the zebrafish embryo

Embryonic development is a complex phenomenon that integrates genetic regulation and biomechanical cellular behaviors. However, the relative influence of these factors on spatiotemporal morphogen distributions is not well understood. Bone Morphogenetic Proteins (BMPs) are the primary morphogens guiding the dorsal-ventral (DV) patterning of the early zebrafish embryo, and BMP signaling is regulated by a network of extracellular and intracellular factors that impact the range and signaling of BMP ligands. Recent advances in understanding the mechanism of pattern formation support a source-sink mechanism, however, it is not clear how the source-sink mechanism shapes the morphogen patterns in three-dimensional (3D) space, nor how sensitive the pattern is to biophysical rates and boundary conditions along both the anteroposterior (AP) and DV axes of the embryo, nor how the patterns are controlled over time. Throughout blastulation and gastrulation, major cell movement, known as epiboly, happens along with the BMP-mediated DV patterning. The layer of epithelial cells begins to thin as they spread toward the vegetal pole of the embryo until it has completely engulfed the yolk cell. This dynamic domain may influence the distributions of BMP network members through advection. We developed a Finite Element Model (FEM) that incorporates all stages of zebrafish embryonic development data and solves the advection-diffusion-reaction Partial Differential Equations (PDE) in a growing domain. We use the model to investigate mechanisms in underlying BMP-driven DV patterning during epiboly. Solving the PDE is computationally expensive for parameter exploration. To overcome this obstacle, we developed a Neural Network (NN) metamodel of the 3D embryo that is accurate and fast and provided a nonlinear map between high-dimensional input and output that replaces the direct numerical simulation of the PDEs. From the modeling and acceleration by the NN metamodels, we identified the impact of advection on patterning and the influence of the dynamic expression level of regulators on the BMP signaling network.

A Krüppel-like factor is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians.

Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk cell–generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here, we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells (PGCs), presumptive germline stem cells (GSCs), and yolk cell progenitors. Knockdown of this klf4-like (klf4l) gene results in animals that fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ cell–like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.

FLUORESCEIN LABELLED ANNEXIN V STAINING LIMITATIONS FOR DETERMINATION OF BLASTODERMAL CELLS APOPTOSIS

It is known that blastoderm contains not only blastodermal cells (BCs) but also cellular “debris”. These particles include yolk granules and lipid droplets. Some authors used AnnexinV (AnV) staining technique for determination of cell apoptosis. However, AnV is a protein with high affinity to lipids. So the aim of our study was to demonstrate the AnV binding in the blastoderm and yolk cell suspensions. Cell populations were obtained from fertilized chicken eggs. The blastoderms were isolated using filter paper ring method and mechanically fragmented. After that, samples were stained with AnV-FITC and analyzed by fluorescent microscopy (FM) and flow cytometry (FC). FM showed non-specific binding of AnV-FITC on the yolk and lipid particles. Therefore, the FC method using specific nuclear dye DRAQ5 was used to separate BCs from cellular debris. Only 1.82 ± 0.27 % cells were DRAQ5 positive. On the other hand, increase of total events (21 ± 3.30 %) of AnV positive cells without DRAQ5 dye was recorded. Moreover, the affinity of yolk to AnV by FC was also observed (10.45 ± 2.59 %). Significant differences among the groups (P &lt; 0.001) were recorded. This trend was caused by non-specific binding of AnV to the lipid and yolk particles. So it is necessary to stain suspension with nuclear dye to separate BCs than co-stained with some marker of apoptosis. Based on the results we can summarized that AnV staining is inappropriate technique for apoptosis of BCs, however, using nuclear dye we can prevent of non-specific binding.

Blastomeres derived from the vegetal pole provide extra-embryonic nutrition to sturgeon (Acipenser) embryos: Transition from holoblastic to meroblastic cleavage

Generally, holoblastic cleavage in embryos (as in amphibians) in which all blastomeres contribute to one of the germ layers, are preserved as a stem lineage of vertebrates, and meroblastic cleavage has evolved independently in each vertebrate lineage. The increasing egg size: yolk volume is the key factor for transition from holoblastic to meroblastic cleavage patterns. Sturgeon (Acipenser) eggs are two times larger than those of the African clawed frog Xenopus laevis (amphibian); despite the varying size, sturgeon embryos retain nearly the same developmental characteristics as X. laevis. Comparatively, the fate of blastomeres derived from the vegetal pole (VP) of a sturgeon embryo is unspecified. Thus, the goal of this study was to determine whether the VP of the embryo contributes to embryonic development, or is simply extra-embryonic. This may also reveal whether the transition of the cleavage pattern (holoblastic to meroblastic) in the actinopterygian lineage is correlated with the egg size: yolk volume. Here, we found that sturgeon vegetal blastomeres formed only primordial germ cells, and the rest were made up of cellular yolk (yolk cells; YCs). Morphological and phenotypic characteristics revealed that after the 1 k-cell / mid-blastula transition, YCs became transcriptionally inactive and served only to provide nutrition to larvae as they developed. Furthermore, inhibition of vegetal blastomeres revealed that sturgeon can utilize their yolk in an acellular form, similar to teleosts, implying that meroblastic cleavage in the actinopterygians, like teleosts, might have evolved by the fusion of the vegetal blastomeres.

Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure

The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. Our patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. Here, we used this tool to quantify the in vivo force production of Myosin II waves that we observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing for the first time to our knowledge quantitative values on an active Myosin-driven force, we elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and we characterized the mechanical properties of the dorsal yolk cell surface.

The Adaptor Protein Lurap1 Is Required for Cell Cohesion during Epiboly Movement in Zebrafish.

Simple SummaryCell adhesion and active cell shape changes play an important role in morphogenetic movements during embryonic development. Zebrafish is an attractive model for the study of cellular and molecular mechanisms underlying these processes. Epiboly is a conserved gastrulation cell movement, which describes the thinning and spreading of an external sheet of cells to cover other groups of cells in the embryo. It involves differential cellular adhesive properties and dynamic cytoskeletal organization across the embryo, but how these are regulated remains elusive. We found that the adaptor protein Lurap1, which interacts with other proteins required for cell migration, plays a role in cell adhesion during epiboly. In zebrafish mutants with loss of Lurap1 function, there is a reduced cellular cohesion in the epithelial blastoderm cells and a delayed epiboly movement. Our observations suggest that Lurap1 is implicated in the regulation of cellular behavior changes for coordinated morphogenetic movements in vertebrate embryos.Cell adhesion and polarized cellular behaviors play critical roles in a wide variety of morphogenetic events. In the zebrafish embryo, epiboly represents an important process of epithelial morphogenesis that involves differential cell adhesion and dynamic cell shape changes for coordinated movements of different cell populations, but the underlying mechanism remains poorly understood. The adaptor protein Lurap1 functions to link myotonic dystrophy kinase-related Rac/Cdc42-binding kinase with MYO18A for actomyosin retrograde flow in cell migration. We previously reported that it interacts with Dishevelled in convergence and extension movements during gastrulation. Here, we show that it regulates blastoderm cell adhesion and radial cell intercalation during epiboly. In zebrafish mutant embryos with loss of both maternal and zygotic Lurap1 function, deep cell multilayer of the blastoderm exhibit delayed epiboly with respect to the superficial layer. Time-lapse imaging reveals that these deep cells undergo unstable intercalation, which impedes their expansion over the yolk cell. Cell sorting and adhesion assays indicate reduced cellular cohesion of the blastoderm. These defects are correlated with disrupted cytoskeletal organization in the cortex of blastoderm cells. Thus, the present results extend our previous works by demonstrating that Lurap1 is required for cell adhesion and cell behavior changes to coordinate cell movements during epithelial morphogenesis. They provide insights for a further understanding of the regulation of cytoskeletal organization during gastrulation cell movements.

Rab5ab-Mediated Yolk Cell Membrane Endocytosis Is Essential for Zebrafish Epiboly and Mechanical Equilibrium During Gastrulation.

Morphogenesis in early embryos demands the coordinated distribution of cells and tissues to their final destination in a spatio-temporal controlled way. Spatial and scalar differences in adhesion and contractility are essential for these morphogenetic movements, while the role that membrane remodeling may play remains less clear. To evaluate how membrane turnover modulates tissue arrangements we studied the role of endocytosis in zebrafish epiboly. Experimental analyses and modeling have shown that the expansion of the blastoderm relies on an asymmetry of mechanical tension in the yolk cell generated as a result of actomyosin-dependent contraction and membrane removal. Here we show that the GTPase Rab5ab is essential for the endocytosis and the removal of the external yolk cell syncytial layer (E-YSL) membrane. Interfering in its expression exclusively in the yolk resulted in the reduction of yolk cell actomyosin contractility, the disruption of cortical and internal flows, a disequilibrium in force balance and epiboly impairment. We conclude that regulated membrane remodeling is crucial for directing cell and tissue mechanics, preserving embryo geometry and coordinating morphogenetic movements during epiboly.

FLUORESCEIN LABELLED ANNEXIN V STAINING LIMITATIONS FOR DETERMINATION OF BLASTODERMAL CELLS APOPTOSIS

It is known that blastoderm contains not only blastodermal cells (BCs) but also cellular “debris”. These particles include yolk granules and lipid droplets. Some authors used AnnexinV (AnV) staining technique for determination of cell apoptosis. However, AnV is a protein with high affinity to lipids. So the aim of our study was to demonstrate the AnV binding in the blastoderm and yolk cell suspensions. Cell populations were obtained from fertilized chicken eggs. The blastoderms were isolated using filter paper ring method and mechanically fragmented. After that, samples were stained with AnV-FITC and analyzed by fluorescent microscopy (FM) and flow cytometry (FC). FM showed non-specific binding of AnV-FITC on the yolk and lipid particles. Therefore, the FC method using specific nuclear dye DRAQ5 was used to separate BCs from cellular debris. Only 1.82 ± 0.27 % cells were DRAQ5 positive. On the other hand, increase of total events (21 ± 3.30 %) of AnV positive cells without DRAQ5 dye was recorded. Moreover, the affinity of yolk to AnV by FC was also observed (10.45 ± 2.59 %). Significant differences among the groups (P < 0.001) were recorded. This trend was caused by non-specific binding of AnV to the lipid and yolk particles. So it is necessary to stain suspension with nuclear dye to separate BCs than co-stained with some marker of apoptosis. Based on the results we can summarized that AnV staining is inappropriate technique for apoptosis of BCs, however, using nuclear dye we can prevent of non-specific binding.