Gluconeogenesis in the extraembryonic yolk syncytial layer of the zebrafish embryo
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PROVIDER: S-EPMC10997050 | biostudies-literature | 2024 Mar
REPOSITORIES: biostudies-literature
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Project description:The yolk syncytial layer (YSL) performs multiple critical roles during zebrafish development. However, little is known about the cellular and molecular mechanisms that underlie the formation of this important extraembryonic structure. Here, we demonstrate by timelapse confocal microscopy of a transgenic line expressing membrane-targeted GFP that the YSL forms as a result of the absence of cytokinesis between daughter nuclei at the tenth mitotic division and the regression of pre-existing marginal cell membranes, thus converting the former margin of the blastoderm into a syncytium. We show that disruption of components of the cytoskeleton induces the formation of an expanded YSL, and identify Rock1 as the regulator of cytoskeletal dynamics that lead to YSL formation. Our results suggest that the YSL forms as a result of controlled cytokinesis failure in the marginal blastomeres, and Rock1 function is necessary for this process to occur. Uncovering the cellular and molecular mechanisms underlying zebrafish YSL formation offers significant insight into syncytial development in other tissues as well as in pathological conditions.
| S-EPMC3507218 | biostudies-literature
Project description:The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation.
| S-EPMC3058998 | biostudies-literature
Project description:The yolk syncytial layer (YSL) in the zebrafish embryo is a multinucleated syncytium essential for embryo development, but the molecular mechanisms underlying YSL formation remain largely unknown. Here we show that zebrafish solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL and that slc3a2 knockdown causes severe YSL defects including clustering of the yolk syncytial nuclei and enhanced cell fusion, accompanied by disruption of microtubule networks. Expression of a constitutively active RhoA mimics the YSL phenotypes caused by slc3a2 knockdown, whereas attenuation of RhoA or ROCK activity rescues the slc3a2-knockdown phenotypes. Furthermore, slc3a2 knockdown significantly reduces tyrosine phosphorylation of c-Src, and overexpression of a constitutively active Src restores the slc3a2-knockdown phenotypes. Our data demonstrate a signaling pathway regulating YSL formation in which Slc3a2 inhibits the RhoA/ROCK pathway via phosphorylation of c-Src to modulate YSL microtubule dynamics. This work illuminates processes at a very early stage of zebrafish embryogenesis and more generally informs the mechanism of cell dynamics during syncytium formation.
| S-EPMC3295254 | biostudies-literature
Project description:Na(+),K(+) ATPase pumps Na(+) out of and K(+) into the cytosol, maintaining a resting potential that is essential for the function of excitable tissues like cardiac muscle. In addition to its well-characterized physiological role in the heart, Na(+),K(+) ATPase also regulates the morphogenesis of the embryonic zebrafish heart via an as yet unknown mechanism. Here, we describe a novel non-cell autonomous function of Na(+),K(+) ATPase/Atp1a1 in the elongation of the zebrafish heart tube. Embryos lacking Atp1a1 function exhibit abnormal migration behavior of cardiac precursors, defects in the elongation of the heart tube, and a severe reduction in ECM/Fibronectin deposition around the myocardium, despite the presence of normal cell polarity and junctions in the myocardial epithelium prior to the timeframe of heart tube elongation. Interestingly, we found that Atp1a1 is not present in the myocardium at the time when cardiac morphogenesis defects first become apparent, but is expressed in an extra-embryonic tissue, the yolk syncytial layer (YSL), at earlier stages. Knockdown of Atp1a1 activity specifically in the YSL using morpholino oligonucleotides produced heart tube elongation defects like those found in atp1a1 mutants, indicating that Atp1a1 function in the YSL is necessary for heart tube elongation. Furthermore, atp1a1 expression in the YSL was regulated by the homeobox transcription factor mxtx1. Together, these data reveal a new non-cell autonomous role for Atp1a1 in cardiac morphogenesis and establish Na(+),K(+) ATPase as a major player in the genetic pathway by which the YSL regulates embryonic ECM deposition.
| S-EPMC3265686 | biostudies-literature
Project description:The front-end desaturases (Fads) are rate-limiting enzymes responsible for production of long-chain polyunsaturated fatty acids (LC-PUFA). The full spectrum of the transcriptional regulation of fads is still incomplete, as cloning of fads promoter is limited to a few species. Here, we described the cloning and characterisation of the zebrafish fads2 promoter. Using 5'-deletion and mutation analysis on this promoter, we identified a specific region containing the sterol regulatory element (SRE) which is responsible for the activation of the fads2 promoter. In tandem, two conserved CCAAT boxes were also present adjacent to the SRE and mutation of either of these binding sites attenuates the transcriptional activation of the fads2 promoter. An in vivo analysis employing GFP reporter gene in transiently transfected zebrafish embryos showed that this 1754 bp upstream region of the fads2 gene specifically directs GFP expression in the yolk syncytial layer (YSL) region. This indicates a role for LC-PUFA in the transport of yolk lipids through this tissue layer. In conclusion, besides identifying novel core elements for transcriptional activation in zebrafish fads2 promoter, we also reveal a potential role for fads2 or LC-PUFA in YSL during development.
| S-EPMC5832746 | biostudies-literature
Project description:Yolk sac membranes of layer eggs were collected daily (n = 7-9) from day three of incubation to day three post-hatch, and mRNA expression and activities were quantified for key gluconeogenesis enzymes (glucose-6-phosphatase, fructose-1,6-bisphosphatase, cytosolic and mitochondrial phosphoenolpyruvate carboxykinases, and pyruvate carboxylase). Lactate, triglycerides, non-esterified fatty acids, glycogen, and glucose in the yolk sac membrane, and blood glucose levels were also measured. The mRNA expression and activity were detected for all enzymes. Differences in expression levels and enzyme activities seemed to reflect the embryo's developmental environment and physiological demands at different developmental stages. During the first week to the mid-second week of incubation, the expression and activity of gluconeogenic enzymes and lactate concentrations were high, suggesting an active period of gluconeogenesis from lactate, reflecting possible hypoxia in the embryo before completed formation of the chorioallantoic capillaries. From the mid-second week to mid-third week, when embryos were in an aerobic state, the triglyceride and non-esterified fatty acid contents increased in the yolk sac. Triglycerides from yolk lipids are typically hydrolyzed to produce non-esterified fatty acids as an energy source, whereas the glycerol skeleton is used for gluconeogenesis. In the late third week, when embryos were considered to re-enter an anaerobic state, the mRNA expression and enzyme activity of only glucose-6-phosphatase were high and the amount of glycogen in the yolk sac was reduced. Therefore, it is suggested that gluconeogenesis activity is low during this period, and the carbohydrates stored in the yolk sac membrane are secreted into the blood as energy for hatching. This study confirmed the role of the yolk sac membrane as a vital gluconeogenic organ during chicken egg incubation.
| S-EPMC10406515 | biostudies-literature
Project description:BackgroundThe number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish.ResultsExpression of a gene encoding the retinol binding protein 4 (Rbp4) was analyzed in zebrafish. rbp4, which is expressed mainly in the liver in adults, was shown to be expressed in the yolk syncytial layer (YSL) during early embryogenesis. At 12-16 hpf rbp4 expression was restricted to the ventro-lateral YSL and later expanded to cover the posterior YSL. We demonstrated that rbp4 expression was negatively regulated by Nodal and Hedgehog (Hh) signalling and positively controlled by retinoic acid (RA). Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration. Since exocrine pancreas, endocrine pancreas, intestine and heart developed normally in Rbp4 morphants, we suggest that rbp4 expression in the YSL is required only for liver development.ConclusionThe characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature. YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.
| S-EPMC2198918 | biostudies-literature
Project description:During times of maternal stress, developing embryos can be exposed to elevated levels of glucocorticoids, which can affect development and permanently alter offspring phenotype. In placental species, the placenta mediates fetal exposure to maternal glucocorticoids via metabolism, yet the placenta itself responds to glucocorticoids to regulate offspring growth and development. In oviparous species, maternal glucocorticoids can be deposited into the egg yolk and are metabolized early in development. This metabolism is mediated by the extraembryonic membranes, but it is unknown if the extraembryonic membranes also respond to maternal glucocorticoids in a way comparable to the placenta. In this study, we quantified the expression of acyl-CoA thioesterase 13 (Acot13) as an initial marker of the membrane's response to corticosterone in chicken (Gallus gallus) eggs. Acot13 regulates fatty acid processing in the embryo, to potentially regulate resource availability during development. We addressed the following questions using Acot13 expression: 1) Do the extraembryonic membranes respond to yolk corticosterone early in development? 2) Is the response to corticosterone dependent on the dose of corticosterone? 3) What is the duration of the response to corticosterone? 4) Does a metabolite of corticosterone (5β-corticosterone) elicit the same response as corticosterone? We found that corticosterone significantly induces the expression of Acot13 on day four of development and that expression of Acot13 increases with the dose of corticosterone. Further, we found expression of Acot13 is significantly elevated by corticosterone on days four and six of development compared to oil treated eggs, but not on days eight and ten. Although this response is transient, it occurs during a critical period of development and could initiate a cascade of events that ultimately alter offspring phenotype. Finally, we found that 5β-corticosterone does not increase the expression of Acot13, indicating that metabolism inactivates corticosterone. Ultimately, this study provides insight into the mechanisms underlying how maternally deposited glucocorticoids can affect embryonic development.
| S-EPMC10836647 | biostudies-literature
Project description:BackgroundThe signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. Several signaling pathways, including Notch and VEGF signaling, are critical for the formation of the vasculature in the mouse. To further understand the role of Notch signaling during endothelial differentiation and the genes regulated by this pathway, both loss-of-function and gain-of-function approaches were analyzed in vivo.ResultsConditional transgenic models were used to expand and ablate Notch signaling in the early embryonic endothelium. Embryos with activated Notch1 signaling in the vasculature displayed a variety of defects, and died soon after E10.5. Most notably, the extraembryonic vasculature of the yolk sac displayed remodeling differentiation defects, with greatly enlarged lumens. These phenotypes were distinct from endothelial loss-of-function of RBPJ, a transcriptional regulator of Notch activity. Gene expression analysis of RNA isolated from the yolk sac endothelia of transgenic embryos indicated aberrant expression in a variety of genes in these models. In particular, a variety of secreted factors, including VEGF and TGF-β family members, displayed coordinate expression defects in the loss-of-function and gain-of-function models.ConclusionsMorphological analyses of the in vivo models confirm and expand the understanding of Notch signaling in directing endothelial development, specifically in the regulation of vessel diameter in the intra- and extraembryonic vasculature. Expression analysis of these in vivo models suggests that the vascular differentiation defects may be due to the regulation of key genes through the Notch-RBPJ signaling axis. A number of these genes regulated by Notch signaling encode secreted factors, suggesting that Notch signaling may mediate remodeling and vessel diameter in the extraembryonic yolk sac via autocrine and paracrine cell communication. We propose a role for Notch signaling in elaborating the microenvironment of the nascent arteriole, suggesting novel regulatory connections between Notch signaling and other signaling pathways during endothelial differentiation.
| S-EPMC3051915 | biostudies-literature
Project description:BackgroundMany species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).ResultsWe hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent.ConclusionWe have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.
| S-EPMC2873407 | biostudies-literature