Double yolk eggs detection using fuzzy logic.

Chicken egg products increased by 60% worldwide resulting in the farmers or traders egg industry. The double yolk (DY) eggs are priced higher than single yolk (SY) eggs around 35% at the same size. Although, separating DY from SY will increase more revenue but it has to be replaced at the higher cost from skilled labor for sorting. Normally, the separation of double yolk eggs required the expertise person by weigh and shape of egg but it is still high error. The purpose of this research is to detect double-yolked (DY) chicken eggs with weight and ratio of the egg's size using fuzzy logic and developing a low cost prototype to reduce the cost of separation. The K-means clustering is used for separating DY and SY, firstly. However, the error from this technique is still high as 15.05% because of its hard clustering. Therefore, the intersection zone scattering from using the weight and ratio of the egg's size to input of DY and SY is taken into consider with fuzzy logic algorithm, to improve the error. The results of errors from fuzzy logic are depended with input membership functions (MF). This research selects triangular MF of weight as low = 65 g, medium = 75 g and high = 85 g, while ratio of the egg is triangular MF as low = 1.30, medium = 1.40 and high = 1.50. This algorithm is not provide the minimum total error but it gives the low error to detect a double yolk while the real egg is SY as 1.43% of total eggs. This algorithm is applied to develop a double yolk egg detection prototype with Mbed platform by a load cell and OpenMV CAM, to measure the weight and ratio of the egg respectively.

Ultrastructural analysis of the yolk processing pattern in embryonic pond slider turtles (Trachemys scripta: Emydidae).

Evolution of the large-yolked, amniotic egg required mechanisms by which extracellular yolk could be made available for embryonic development. In birds, the endodermal lining of the yolk sac absorbs and digests the yolk. In contrast, recent studies on lizards and snakes (squamates) have revealed that yolk is processed by means of a proliferating mass of "spaghetti-like" strands formed by endodermal cells attached to anastomosing blood vessels. To clarify the method of yolk processing in chelonians, we applied electron microscopy to an extensive series of embryos of the pond slider turtle, Trachemys scripta. Our findings demonstrate that proliferating endodermal cells phagocytose yolk spheres. These cells remain attached to one another following mitosis, thereby forming clumps that progressively occupy the yolk sac cavity. Upon invasion of blood vessels, the cells become organized into elongated, vascularized "spaghetti-like" strands of cells like those found in squamates. Residual yolk found in the body cavity of new hatchlings chiefly consists of these vascularized strands. Such strands of cells also develop in the false map turtle, Graptemys pseudographica (Emydidae). We infer that the developmental pattern by which yolk is processed is ancestral for both Chelonia and Reptilia, and therefore must have been modified or abandoned in birds or their archosaur ancestors.

Embryonic and larval development of the northern pike: An emerging fish model system for evo-devo research.

The northern pike, Esox lucius, is one of the largest temperate freshwater apex predators with a characteristic morphology: an elongated body with pelvic, dorsal, and anal fins located at the rear as a functional feature to sprint predation. However, the typical pike character is its head, which is characterized by a long, flattened snout, a well-armed mouth with numerous teeth, and large eyes characteristic of shallow water visual predators. Although the northern pike is becoming increasingly popular as a model system for ecology and evolutionary research, a detailed staging table has not yet been reported. In this study, we report the first comprehensive staging table for the northern pike, spanning from the one-cell stage to the freely-swimming juvenile stage. In addition to classical embryological descriptions, we use a DAPI staining to distinguish individual cells and embryonic structures during the early development. This dataset, in combination with the genomic and transcriptomic resources already available, serves as a foundation for in-depth mechanistic studies dealing with development using this species.

A Novel Pattern of Yolk Processing in Developing Snake Eggs (Colubridae: Lampropeltini) and its Functional and Evolutionary Implications.

Early amniotic vertebrates evolved large-yolked eggs that permitted production of well-developed, terrestrial hatchlings. This reproductive pattern required new mechanisms for cellularizing the yolk and mobilizing it for embryonic use. In birds, cells that line the yolk sac cavity phagocytose and digest the yolk material, a pattern that is commonly assumed to be universal among oviparous amniotes. However, recent evidence challenges the assumption that all squamate reptiles conform to the avian developmental pattern. In this paper, scanning electron microscopy and histology were used to study mechanisms of yolk processing in two colubrid snakes, the kingsnake Lampropeltis getula and the milksnake L. triangulum. Endodermal cells from the yolk sac splanchnopleure proliferate massively as they invade the yolk sac cavity, forming elaborate chains of interlinked cells. These cells grow in size as they phagocytose yolk material. Subsequently, vitelline capillaries invade the masses of yolk-laden cells and become coated with the endodermal cells, forming an elaborate meshwork of cell-coated strands. The close association of cells, yolk, and blood vessels allows yolk material to be cellularized, digested, and transported for embryonic use. The overall pattern is like that of the corn snake Pantherophis guttatus, but contrasts markedly with that of birds. Given recent evidence that this developmental pattern may also occur in certain lizards, we postulate that it is ancestral for squamates. Studies of lizards, crocodilians, and turtles are needed to clarify the evolutionary history of this pattern and its implications for the evolution of the amniotic (terrestrial) vertebrate egg.

Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development.

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation.

Endophilin B is required for the Drosophila oocyte to endocytose yolk downstream of Oskar.

The nutritional environment is crucial for Drosophila oogenesis in terms of controlling hormonal conditions that regulate yolk production and the progress of vitellogenesis. Here, we discovered that Drosophila Endophilin B (D-EndoB), a member of the endophilin family, is required for yolk endocytosis as it regulates membrane dynamics in developing egg chambers. Loss of D-EndoB leads to yolk content reduction, similar to that seen in yolkless mutants, and also causes poor fecundity. In addition, mutant egg chambers exhibit an arrest at the previtellogenic stage. D-EndoB displayed a crescent localization at the oocyte posterior pole in an Oskar-dependent manner; however, it did not contribute to pole plasm assembly. D-EndoB was found to partially colocalize with Long Oskar and Yolkless at the endocytic membranes in ultrastructure analysis. Using an FM4-64 dye incorporation assay, D-EndoB was also found to promote endocytosis in the oocyte. When expressing the full-length D-endoB(FL) or D-endoB(ΔSH3) mutant transgenes in oocytes, the blockage of vitellogenesis and the defect in fecundity in D-endoB mutants was restored. By contrast, a truncated N-BAR domain of the D-EndoB only partially rescued these defects. Taken together, these results allow us to conclude that D-EndoB contributes to the endocytic activity downstream of Oskar by facilitating membrane dynamics through its N-BAR domain in the yolk uptake process, thereby leading to normal progression of vitellogenesis.

Calcium signaling in extraembryonic domains during early teleost development.

It is becoming recognized that the extraembryonic domains of developing vertebrates, that is, those that make no cellular contribution to the embryo proper, act as important signaling centers that induce and pattern the germ layers and help establish the key embryonic axes. In the embryos of teleost fish, in particular, significant progress has been made in understanding how signaling activity in extraembryonic domains, such as the enveloping layer, the yolk syncytial layer, and the yolk cell, might help regulate development via a combination of inductive interactions, cellular dynamics, and localized gene expression. Ca(2+) signaling in a variety of forms that include propagating waves and standing gradients is a feature found in all three teleostean extraembryonic domains. This leads us to propose that in addition to their other well-characterized signaling activities, extraembryonic domains are well suited (due to their relative stability and continuity) to act as Ca(2+) signaling centers and conduits.

Characterization of Ca(2+) signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development.

Preferential loading of the complementary bioluminescent (f-aequorin) and fluorescent (Calcium Green-1 dextran) Ca(2+) reporters into the yolk syncytial layer (YSL) of zebrafish embryos, revealed the generation of stochastic patterns of fast, short-range, and slow, long-range Ca(2+) waves that propagate exclusively through the external YSL (E-YSL). Starting abruptly just after doming (~4.5h post-fertilization: hpf), and ending at the shield stage (~6.0hpf) these distinct classes of waves propagated at mean velocities of ~50 and ~4µm/s, respectively. Although the number and pattern of these waves varied between embryos, their initiation site and arcs of propagation displayed a distinct dorsal bias, suggesting an association with the formation and maintenance of the nascent dorsal-ventral axis. Wave initiation coincided with a characteristic clustering of YSL nuclei (YSN), and their associated perinuclear ER, in the E-YSL. Furthermore, the inter-YSN distance (IND) appeared to be critical such that Ca(2+) wave propagation occurred only when this was <~8µm; an IND >~8µm was coincidental with wave termination at shield stage. Treatment with the IP3R antagonist, 2-APB, the Ca(2+) buffer, 5,5'-dibromo BAPTA, and the SERCA-pump inhibitor, thapsigargin, resulted in a significant disruption of the E-YSL Ca(2+) waves, whereas exposure to the RyR antagonists, ryanodine and dantrolene, had no significant effect. These findings led us to propose that the E-YSL Ca(2+) waves are generated mainly via Ca(2+) release from IP3Rs located in the perinuclear ER, and that the clustering of the YSN is an essential step in providing a CICR pathway required for wave propagation. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Solute carrier family 3 member 2 (Slc3a2) controls yolk syncytial layer (YSL) formation by regulating microtubule networks in the zebrafish embryo.

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.

Dissociation of chicken blastoderm for examination of apoptosis and necrosis by flow cytometry.

We describe a method of isolating blastodermal cells for characterization through flow cytometry. Particular attention was placed on cell viability and integrity issues faced by conventional protocols. The method allowed us to examine mechanisms behind cellular death. Our protocol was optimized by the spatial resolution of the ImageStream multispectral imaging flow cytometer. Overall, the technique provides both quantitative and qualitative information on blastodermal cells. The methodology was applied to the current biological problem in which prolonged (14 d) versus short-term (4 d) layer egg storage reduces embryo viability. Data were obtained between 3 egg storage treatments (unstored, 4 d, and 14 d); the data were analyzed by the PROC MIXED model procedure of SAS at P < or = 0.05 and least squares means separated by the PDIFF procedure of SAS. The results showed that egg storage increases the rate of cell death by both apoptosis and necrosis. Importantly, our study showed higher percentages of necrosis and late apoptosis in long-term versus short-term stored eggs. The percentage of live cells decreased significantly when eggs were stored for 14 d (71.42 + or - 3.36%) compared with eggs stored for 4 d (83.58 + or - 2.15%). The percentage of early apoptotic cells was not significantly different between the 2 treatments. The percentage of necrotic cells and late apoptotic-necrotic cells was higher in eggs stored for 14 d (16.75 + or - 1.73%; 7.36 + or - 1.53%) versus eggs stored for 4 d (3.56 + or - 1.64%; 2.31 + or - 1.52%), respectively. This could negatively affect embryo survival because of the potential effect that necrosis has on surrounding tissue integrity. The technique will be particularly relevant in studies requiring single cells from chicken blastoderms or as a basis to characterize genes that regulate apoptosis in avian species.

Calcium- and polyphosphate-containing acidocalcisomes in chicken egg yolk.

BACKGROUND INFORMATION: Poly P (inorganic polyphosphate) is a polymer formed by P(i) residues linked by high-energy phosphoanhydride bonds. The presence of poly P in bacteria, fungi, algae and protists has been widely recognized, but the distribution of poly P in more complex eukaryotes has been poorly studied. Poly P accumulates, together with calcium, in acidic vesicles or acidocalcisomes in a number of organisms and possesses a diverse array of functions, including roles in stress response, blood clotting, inflammation, calcification, cell proliferation and apoptosis. RESULTS: We report here that a considerable amount of phosphorus in the yolk of chicken eggs is in the form of poly P. DAPI (4',6-diamidino-2-phenylindole) staining showed that poly P is localized mainly in electron-dense vesicles located inside larger vacuoles (compound organelles) that are randomly distributed in the yolk. These internal vesicles were shown to contain calcium, potassium, sodium, magnesium, phosphorus, chlorine, iron and zinc, as detected by X-ray microanalysis and elemental mapping. These vesicles stain with the acidophilic dye Acridine Orange. The presence of poly P in organellar fractions of the egg yolk was evident in agarose gels stained with Toluidine Blue and DAPI. Of the total phosphate (Pi) of yolk organelles, 16% is present in the form of poly P. Total poly P content was not altered during the first 4 days of embryogenesis, but poly P chain length decreased after 1 day of development. CONCLUSIONS: The results of the present study identify a novel organelle in chicken egg yolk comprising acidic vesicles with a morphology, physiology and composition similar to those of acidocalcisomes, within larger acidic vacuoles. The elemental composition of these acidocalcisomes is proportionally similar to the elemental composition of the yolk, suggesting that most of these elements are located in these organelles, which might be an important storage compartment in eggs.

Structural heterogeneity of yolk spheres in hierarchical follicles from hen ovary: a histochemical study.

1. Four types of yolk spheres with variable structure, chemical composition and frequency of occurrence in yolk plasma of hierarchical follicles (F(4), F(3), F(2) and F(1) with diameters of 10.0, 15.5, 20.0 and 35.0 mm, respectively) of the hen ovary were identified using histochemical methods for localising lipids, carbohydrates and proteins. 2. Yolk spheres of the first type (YS(1)) had a phospholipoprotein membrane surrounding fluid matrix which stained lightly for phospholipids, proteins and acidic mucopolysaccharides. Two types of droplets were observed in the matrix of YS(1). Spheres of the second type (YS(2)) had a lipoprotein- and acidic mucopolysaccharide-rich peripheral region and a single large droplet in its fluid matrix. Droplets of YS(2), unlike YS(1), showed three regions and metachromatic staining with ninhydrin-Schiff reagent. The third type of sphere (YS(3)) had a homogeneous matrix staining for proteins, neutral lipids and florescent yellow with alcian blue and differentially with ninhydrin-Schiff reagent; it was bounded by a phospholipids- and acidic mucopolysaccharide-containing thick peripheral region. Its fluid matrix also showed toluidine-blue-positive, densely packed granules and small droplets. The fourth type (YS(4)) was seen only in bromophenol blue and Nile blue preparations, revealing the presence of proteins and neural lipids in their matrix and peripheral regions. 3. Quantitative data on the relative abundance of yolk spheres in F(4) to F(1) follicles revealed more YS(3) (51.1 to 64.7%) than YS(1) (16.2 to 28.3%) and YS(2) (19.1 to 23.2%). The percentage of YS(1) increased and that of YS(3) decreased as follicle size increased.

Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase.

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.

Cloning and expression of carp cathepsin Z: possible involvement in yolk metabolism.

A cDNA encoding cathepsin Z (CTPZ) was cloned from a carp ovarian cDNA library. It is homologous to mammalian CTPZ. The amino acid residues important for protein folding and enzymatic activity of mammalian CTPZ are conserved in carp CTPZ. It is widely expressed in a variety of carp tissues as revealed by Western blot and reverse transcription-polymerase chain reaction. The CTPZ mRNA was transiently accumulated during oocyte maturation. In oocytes, CTPZ is localized in cortical granules and in the cytoplasm surrounding the yolk granules. After fertilization, CTPZ remained associated with the yolk granules while the cortical granular CTPZ was discharged to plasma membrane, perivitelline space, and fertilization envelope. Carp cathepsin Z has proteolytic activity toward vitellogenin that could be inhibited by inhibitors specific for the proteases of papain family. The potential roles of cathepsin Z in carp eggs are discussed.

Reorganization of cytoplasm in the zebrafish oocyte and egg during early steps of ooplasmic segregation.

The aim of this work is to determine when and how ooplasmic segregation is initiated in the zebrafish egg. To this end, the organization of the ooplasm and vitelloplasm were examined in oocytes and eggs shortly after activation. Ooplasmic segregation, initiated in the stage V oocyte, led to the formation of ooplasmic domains rich in organelles, and ribonucleoproteins. A linear array of closely arranged peripheral yolk globules separated an outer domain of ectoplasm from an inner domain of interconnected endoplasmic lacunae. The structure of this yolk array and the distribution of microinjected labeled tracers suggests that it may provide a barrier limiting ooplasm transit. Loosely arranged yolk globules at the animal hemisphere allow wide connections between the endoplasm and a preblastodisc domain. Activation caused further segregation of ooplasm, reorganization of endoplasmic lacunae, and blastodisc growth. The presence of an endoplasmic cytoskeleton suggests that these changes may be driven by microtubules and microfilaments.

Expression of ornithine-urea cycle enzymes in early life stages of air-breathing walking catfish Clarias batrachus and induction of ureogenesis under hyper-ammonia stress.

The air-breathing walking catfish Clarias batrachus is a potential ureogenic teleost with having a full complement of ornithine-urea cycle (OUC) enzymes expressed in various tissues. The present study was aimed at determining the pattern of nitrogenous waste excretion in the form of ammonia-N and urea-N along with the changes of tissue ammonia and urea levels, and the expression of OUC enzymes and glutamine synthetase (GSase) in early life stages of this teleost, and further, to study the possible induction of ureogenesis in 15-day old fry under hyper-ammonia stress. The ammonia and urea excretion was visible within 12 h post-fertilization (hpf), which increased several-fold until the yolk was completely absorbed by the embryo. Although all the early developing stages were primarily ammoniotelic, they also excreted significant amount of nitrogen (N) in the form of urea-N (about 35-40% of total N). Tissue levels of ammonia and urea also increased along with subsequent developmental stages at least until the yolk absorption stage. All the OUC enzymes and GSase were expressed within 4-12 hpf showing an increasing trend of activity for all the enzymes until 350 hpf. There was a significant increase of activity of GSase, carbamyl phosphate synthetase III (CPSase III) and argininosuccinate lyase enzymes (ASL), accompanied with significant increase of enzyme protein concentration of at least two enzymes (GSase and CPSase III) in the 15-day old fry following exposure to 10 mM NH4Cl as compared to respective controls kept in water over a period of 72 h. Thus, it appears that the OUC enzymes are expressed in early life stages of walking catfish like other teleosts, but at relatively high levels and remain expressed all through the life stages with a potential of stimulation of ureogenesis throughout the life cycle as a sort of physiological adaptation to survive and breed successfully under hyper-ammonia and various other environmental-related stresses.

Yolk granule tethering: a role in cell resealing and identification of several protein components.

Homotypic fusion among echinoderm egg yolk granules has previously been reconstituted in vitro, and shown to be a rapid, Ca2+-triggered reaction that can produce extremely large (>10 microm diameter) fusion products. We here show that, prior to Ca2+-triggered fusion, yolk granules in vitro, if isolated in an appropriate buffer, became tethered to one another, forming large aggregates of more than 100 granules. Granule washing with mildly chaotropic salt abolished this tethering reaction, and prevented Ca2+-triggered formation of the large fusion products characteristic of tethered granules. Protein factors present in the wash restored tethering activity and these factors could be substantially enriched by anion exchange chromatography. The enriched fraction behaved under native conditions as a high molecular weight (approximately 670 kDa), multisubunit complex of at least seven proteins. Monoclonal antibodies directed against this complex of proteins were capable of immunodepleting tethering activity, confirming the role of the complex in granule tethering. These antibodies selectively stained the surface of yolk granules in the intact egg. We therefore propose a new role for tethering: it can promote the formation of large vesicular fusion products, such as those required for successful resealing. We have, moreover, identified several proteins that may be critical to this tethering mechanism.

[The study of protoporphyrin IX metabolism in cell proliferation using yolk as the target samples].

In order to find the better target sample to investigate protoporphyrin IX metabolism in the cell proliferation process in chick embryos, the emission spectra of egg white, yolk and urine from non-incubated and incubated eggs were measured under the excitation at 405 nm. It was observed that, among the three components investigated, only yolk sample was possible to get in pureness almost through the whole incubation process. Furthermore, compared with the egg white, the yolk had more obvious characteristic emission of protoporphyrin IX and lower background spectra and turned to be the better target sample for the investigation. The relative characteristic emission intensity of PpIX of yolk samples was used to determine the level of PpIX metabolism. It was found that the relative emission intensity increases during the first 10 days of proliferation process, indicating an increase in the concentration or in the metabolism level of protoporphyrin IX. The relative emission intensity, hence the PpIX metabolism level, then decreased gradually during the rest days of proliferation process. These results are similar to those obtained on human malignant tumors as they develop. The result testifies the point of view further that the abnormity of PpIX metabolism is correlative to the rapid limitless cell proliferation.

Regulation of hhex expression in the yolk syncytial layer, the potential Nieuwkoop center homolog in zebrafish.

The Nieuwkoop center is the earliest signaling center during dorsal-ventral pattern formation in amphibian embryos and has been implied to function in induction of the Spemann-Mangold organizer. In zebrafish, Nieuwkoop-center-like activity resides in the dorsal yolk syncytial layer (YSL) at the interface of the vegetal yolk cell and the blastoderm. hex homologs are expressed in the anterior endomesoderm in frogs (Xhex), the anterior visceral endoderm in mice, and the dorsal YSL in zebrafish (hhex). Here, we investigate the control of hhex expression in the YSL. We demonstrate that bozozok (boz) is absolutely required for early hhex expression, while overexpression of boz causes ectopic hhex expression. Activation of Wnt/beta-catenin signaling by LiCl induces hhex expression in wild-type YSL but not in boz mutant embryos, revealing that boz activity is required downstream of Wnt/beta-catenin signaling for hhex expression. Further, we show that the boz-mediated induction of hhex is independent of the Boz-mediated repression of bmp2b. Our data reveal that repressive effects of both Vega1 and Vega2 may be responsible for the exclusion of hhex expression from the ventral and lateral parts of the YSL. In summary, zebrafish hhex appears to be activated by Wnt/beta-catenin in the dorsal YSL, where Boz acts in a permissive way to limit repression of hhex by Vega1 and Vega2.