Destabilization of vitelline membrane outer layer protein 1 homolog (VMO1) by C-mannosylation.

C-mannosylation is a rare type of protein glycosylation whereby a single mannose is added to the first tryptophan in the consensus sequence Trp-Xaa-Xaa-Trp/Cys (in which Xaa represents any amino acid). Its consensus sequence is mainly found in proteins containing a thrombospondin type-1 repeat (TSR1) domain and in type I cytokine receptors. In these proteins, C-mannosylation affects protein secretion, intracellular localization, and protein stability; however, the role of C-mannosylation in proteins that are not type I cytokine receptors and/or do not contain a TSR1 domain is less well explored. In this study, we focused on human vitelline membrane outer layer protein 1 homolog (VMO1). VMO1, which possesses two putative C-mannosylation sites, is a 21-kDa secreted protein that does not contain a TSR1 domain and is not a type I cytokine receptor. Mass spectrometry analyses revealed that VMO1 is C-mannosylated at Trp105 but not at Trp44 . Although C-mannosylation does not affect the extracellular secretion of VMO1, it destabilizes the intracellular VMO1. In addition, a structural comparison between VMO1 and C-mannosylated VMO1 showed that the modification of the mannose changes the conformation of three loops in VMO1. Taken together, our results demonstrate the first example of C-mannosylation for protein destabilization of VMO1.

Pacific white shrimp (Litopenaeus vannamei) vitelline membrane outer layer protein 1 (VMO1) is produced in the hepatopancreas and transported into ovarian oocytes during vitellogenesis.

In birds, vitelline membrane outer layer protein 1 (VMO1) is an exogenous protein that can be absorbed by eggs as a barrier to prevent the mixing of yolk and egg white. However, researches on VMO1 are limited in birds but not other non-avian species until now. In this study, we first identified a novel Vmo1 cDNA (Lv-Vmo1) in Pacific white shrimp (Litopenaeus vannamei), the most important cultured shrimp in the world. We further analyzed its gene organization, phylogenetic relationship and protein structure. The Lv-Vmo1 transcript was specifically expressed in the hepatopancreas without sexual dimorphism. During ovarian development in female, the hepatopancreatic Lv-Vmo1 mRNA levels showed a significant increase. By in situ hybridization, Lv-Vmo1 mRNA was present in three cell types of the hepatopancreas but neither oocytes nor follicle cells of the ovary. In contrast, immunofluorescence revealed that Lv-VMO1 protein was distributed in the cytoplasms of both hepatopancreatic cells and ovarian oocytes. Western blot showed that Lv-VMO1 protein was produced in the hepatopancreas and transported to the ovary via hemolymph circulation. Identification of a species-specific egg-entry guide protein is the key to the receptor-mediated ovarian transduction of cargo, a novel gene editing approach in oviparous animals. This study lays the mechanism for exogenous transport into penaeid shrimp eggs by VMO1, as a foundation for achieving exogenous protein-mediated incorporation into oocytes.

Molecular characteristics of the edge cells responsible for expansion of the chick embryo on the vitelline membrane.

During early avian development, only a narrow band of cells (the edge cells, also called 'margin of overgrowth') at the rim of the embryo is responsible for blastoderm expansion by crawling over the vitelline membrane (VM) to cover the whole egg yolk in just 4 days (a process called epiboly). Surprisingly, this has not yet been studied in detail. Here we explore the edge cells of the chick embryo using in situ hybridization, immunohistochemistry and live imaging. Morphological and molecular properties reveal that the edge has a distinctive structure, being subdivided into sub-regions, including at least four distinct zones (which we name as leading, trailing, deep and stalk zones). This allows us to study reorganization of the edge region that accompanies reattachment of an explanted blastoderm to the VM. Immunohistochemistry uncovers distinct polarized cellular features resembling the process of collective cell migration described in other systems. Live imaging reveals dynamic lamellipodial and filopodial activity at the leading edge of the outermost cells. Our data provide evidence that edge cells are a distinct tissue. We propose that edge cells may be a useful model system for the study of wound healing and other closure events in epithelial cell sheets.

Vitelline Membrane Protein 26 Mutagenesis, Using CRISPR/Cas9, Results in Egg Collapse in Plutella xylostella.

Vitelline membrane proteins (VMPs) are the main proteins that form the inner shell (vitelline membrane layer) of insect eggs and are an integral part of egg formation and embryo development. Here, we characterized the molecular structure and expression patterns of the VMP26 gene and analyzed its reproductive functions in diamondback moth, Plutella xylostella (L.), a worldwide migratory pest of cruciferous plants. The PxVMP26 gene was shown to be a single exon gene that contained an open reading frame of 852 base pairs (bp) encoding 283 amino acids. Both qPCR and western blot analyses showed that PxVMP26 was specifically expressed in female adults and was significantly highly expressed in the ovary. Further anatomical analysis indicated that the expression level of PxVMP26 in the ovarian tube with an incomplete yolk was significantly higher than that in the ovarian tube with a complete yolk. CRISPR/Cas9-induced PxVMP26 knockout successfully created two homozygous strains with 8- and 46-bp frameshift mutations. The expression deficiency of the PxVMP26 protein was detected in the mutant strains using immunofluorescence and western blot. No significant difference was found in the number of eggs laid within three days between wild and mutant individuals, but there was a lower egg hatchability. The loss of the PxVMP26 gene changed the mean egg size, damaged the structure of the vitelline membrane, and increased the proportion of abnormal eggs due to water loss, resulting in egg collapse. This first analysis of the roles of the VMP gene in the oocyte formation and embryonic development of P. xylostella, using CRISPR/Cas9 technology, provides a basis for screening new genetic control targets of P. xylostella.

Removal of the block to self-fertilization by low-calcium artificial seawater in the ascidian Ciona intestinalis.

Ascidians (Urochordate) are hermaphroditic marine invertebrates that release sperm and eggs to the surrounding seawater. However, several ascidians, including Ciona intestinalis and Halocynthia roretzi, show strict self-sterility due to a self/nonself-recognition mechanism in the interaction between sperm and the vitelline coat (VC) of the eggs. We have previously reported that sperm intracellular Ca2+ level drastically increased immediately after sperm binding to the VC of self eggs but not nonself eggs in C. intestinalis type A, which was potently inhibited by lowering the external Ca2+ concentration, suggesting that sperm Ca2+ influx occurs after sperm self-recognition on the VC. Here, we investigated whether self-sterility was abolished by lowering the external Ca2+ concentration in C. intestinalis. The results showed that the block to self-fertilization was removed by low-Ca2+ (∼1 mM) seawater without decreasing the fertilization rate. Such an effect was not observed with Mg2+ or K+. These results led us to conclude that a low-Ca2+ environment is sufficient to block the self-recognition signal upon fertilization. As low-Ca2+ seawater showed no effect on H. roretzi self-sterility, we propose that the mechanism of self-sterility in Ciona must be distinctive from that in Halocynthia.

Citric acid as a factor limiting changes in the quality of table eggs during their storage.

The aim of the experiment was to evaluate the potential use of citric acid as a modifier of quality changes in table eggs during their storage. About 780 table hen eggs were collected on the same day. They were numbered individually and placed on trays 30 pcs on each. Control group (CA0) consisted of eggs unmodified with any additional substances. In experimental groups CA10 and CA15, eggshells were sprayed with the aqueous solution of citric acid (10 and 15% concentration, respectively). At the start of the experiment, only quality traits of eggs from the control group were analyzed. The remaining eggs were stored at 14°C and 70% RH (typical storage conditions). Their quality was evaluated after 7, 14, 21, and 28 d. The depth of the air cell, egg weight and specific gravity, traits of shell (permeability, strength, weight, thickness, density), and egg content (pH of yolk and albumen, Haugh units, yolk weight and color) were evaluated each time. The use of citric acid decreased the severity of qualitative changes. Citric acid-treated eggs demonstrated smaller weight loss, shallower air cell, higher structural albumen, less-intensive water diffusion from albumen to yolk indicating the improved resistance of the vitelline membrane. Owing to the fact that citric acid is accepted and recognized as a safe food preservative is a relatively cheap and available substance, it seems that it can be used to inhibit quality changes in table eggs during their storage.

Comparative N-Glycoproteomic Analysis Provides Novel Insights into the Deterioration Mechanisms in Chicken Egg Vitelline Membrane during High-Temperature Storage.

The weakening of chicken egg vitelline membrane (CEVM) is one of the most important factors influencing egg quality during high-temperature storage. Therefore, a comparative N-glycoproteomic analysis of CEVM after 10 days of storage at 30 °C was performed to explore the roles of protein N-glycosylation in membrane deterioration. In total, 399 N-glycosites corresponding to 198 proteins were identified, of which 46 N-glycosites from 30 proteins were significantly altered. Gene ontology analysis revealed that these differentially N-glycosylated proteins (DGPs) were involved in antibacterial activity, glycosaminoglycan binding, lipid binding, and aminopeptidase activity. Removal of the N-glycans in Mucin-5B may result in a loss of CEVM's mechanical properties. The N-glycosites enriched in the apolipoprotein B ß2 domain in CEVM were significantly changed, which may contribute to lipid composition modifications during storage. Moreover, N-glycosites in several metalloproteases were located within the functional domain or active site region, indicating that the decreased N-glycosylation levels may affect their structural stability, specific substrate binding, or enzyme activity. These findings provide novel insights into the roles of protein N-glycosylation during membrane weakening.

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

Structural and proteomic analyses of vitelline membrane proteins of blackbird (Turdus merula) and song thrush (Turdus philomelos).

In this study, we aimed to perform structural and proteomic analysis of the vitelline membrane (VM) of two species birds belonging to the family Turdidae: blackbird (Turdus merula) and song thrush (Turdus philomelos). We performed structural analyses using scanning electron microscopy. The VM proteins were identified and compared to the best-known chicken VM proteins. According to our results, VM of both species has a typical three-layered structure: the outer layer, inner layer, and the continuous membrane between them. An unusual observation was the finding of "convexity" formed by the inner layer in blackbird. The role of these convex structures is not known, but they can be typical for the species and can be used in their identification. In addition, we identified two proteins in the VM of both species of birds, of which U3KEZ1 FICAL was not previously identified in any other bird species, and the U3JXV8 FICAL protein was confirmed only once in cockatiel parrot VM. The function of these proteins is not exactly known, but their structure shows similarities to the SERPIN proteins that are involved in microbiological defense, i.e., they are immune proteins. This study contributes to the current knowledge about the structure and composition of proteins of VM, especially because similar analyses have never been performed for Turdidae family. Knowledge of the structure and specific proteins of blackbird and song thrush VM can be beneficial in research on ecology and bird biology and also helpful in developing noninvasive and nongenetic identification methods.

Quantitative Comparative Proteomic Analysis of Chicken Egg Vitelline Membrane Proteins during High-Temperature Storage.

To explore the thermally induced alterations in chicken egg vitelline membrane (CEVM) protein abundances, a comparative proteomic analysis of CEVM after 10 days of storage at 30 °C was performed. Altogether, 981 proteins were identified, of which 124 protein abundances were decreased and 79 were increased. Bioinformatic analysis suggested that the altered proteins were related to structure (n = 10), mechanical properties (n = 13), chaperone (n = 15), antibacterial (n = 12), and antioxidant (n = 3). Alterations in abundances of structural proteins, possibly resulting from the disintegration of these complexes, were observed in this study, suggesting a loss in fibrous structure. Several proteins involved in mechanical strength (n = 10), elasticity (n = 3), and chaperone were decreased in abundances, which indicated that deficits in these proteins might affect the CEVM mechanical properties. These findings will extend our understanding of CEVM deterioration during high-temperature storage from a proteomic perspective.

A puzzle piece of protein N-glycosylation in chicken egg: N-glycoproteome of chicken egg vitelline membrane.

The chicken egg vitelline membrane (CEVM) is an important structure for the transmembrane transport of egg yolk components, protection of the blastodisc, and separation of egg white and egg yolk. In this study, the N-glycoproteome of the CEVM was mapped and analyzed in depth. Total protein of the CEVM was digested, and the glycopeptides were enriched by a hydrophilic interaction liquid chromatography microcolumn and identified by nano liquid chromatography/tandem mass spectrometry. A total of 435 N-glycosylation sites on 208 N-glycoproteins were identified in CEVM. Gene Ontology enrichment analysis showed that CEVM N-glycoproteins are mainly involved in the regulation of proteinases/inhibitors and transmembrane transport of lipids. Mucin-5B is the primary N-glycoprotein in the CEVM. Comparison of the main N-glycoproteins between the CEVM and other egg parts revealed the tissue specificity of N-glycosylation of egg proteins. The results provide insights into protein N-glycosylation in the chicken egg, CEVM functions and underlying mechanisms.

Interrelationships of vitelline and muscle cells within the vitelline follicles of the blood fluke Aporocotyle simplex (Digenea, Aporocotylidae) and morphological evidence for the modification of vitelline material for eggshell formation.

This study of the fish blood fluke Aporocotyle simplex represents the first detailed transmission electron microscopical (TEM) investigation of the vitellarium of an aporocotylid digenean blood fluke. It revealed some unusual characteristics in the cytoarchitecture of the vitelline follicles and demonstrated modifications of the vitelline granules for eggshell formation. The vitelline follicles consist of vitellocytes at different developmental stages surrounded by sarcoplasmic processes of myocytes which occur throughout each follicle. Sites of intimate contact occur between the vitellocytes and the myocytes. Individual vitelline globules (0.1-0.2 µm in diameter) accumulate in quite small clusters of 10-20 and have a dense, heterogeneous matrix possessing central and peripheral regions with a greater density. Modifications of the vitelline globules take place within the clusters and are first apparent when the vitellocytes reach the lumen of the vitelline duct and vitelline reservoir. Globules within the clusters become confluent, and, when the vitellocytes reach the lumen of the oviduct and proximal ootype, these consolidated clusters contain a shapeless, loosely packed, dense material which is released from the vitellocytes by exocytosis. This investigation has provided morphological evidence for shell formation from modified vitelline globules in the form of a discontinuous, thin layer (~ 0.07 µm in thickness) of electron-dense shell material around the fertilized ovum and associated vitellocytes in the proximal ootype. The eggshell of intra-uterine eggs acquires an additional thin, heterogeneous outer layer, increasing its thickness to ~ 0.1 µm. The cytoarchitecture of the vitellarium, modifications of the vitelline globules within the clusters and the structure of the eggshell of A. simplex may prove to be of value in studies examining relationships between the three distinct lineages of aporocotylid digeneans.

Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, domestic pigeon) birds.

Of all the known oviparous taxa, female birds lay the most diverse types of eggs that differ in terms of shape, shell pigmentation, and shell structure. The pigmentation of the shell, the weight of the egg, and the composition of the yolk correlate with environmental conditions and the needs of the developing embryos. In this study, we analyzed the structure and protein composition of the vitelline membrane (VM) of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. We found that the VM structure is characteristic of each species and varies depending on whether the species is precocial (ring-necked pheasant and gray partridge) or superaltrical (cockatiel parrot and domestic pigeon). We hypothesize that a multilayer structure of VM is necessary to counteract the aging process of the egg. The multilayer structure of VM is only found in species with a large number of eggs in one clutch and is characterized by a long incubation period. An interesting discovery of this study is the three-layered VM of pheasant and partridge eggs. This shows that the formation of individual layers of VM in specific sections of the hen's reproductive system is not confirmed in other species. The number of protein fractions varied between 19 and 23, with a molecular weight ranging from 15 to 250 kDa, depending on the species. The number of proteins identified in the VM of the study birds' eggs is as follows: chicken-14, ring-necked pheasant-7, gray partridge-10, cockatiel parrot-6, and domestic pigeon-23. The highest number of species-specific proteins (21) was detected in the VM of domestic pigeon. This study is the first to present the structure and protein composition in the VM of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. In addition, we analyzed the relationship between the hatching specification of birds and the structure of the VM.

Identification of 56 Proteins Involved in Embryo-Maternal Interactions in the Bovine Oviduct.

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4-5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4-6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

Morphometric analysis of the branching of the vascular tree in the chick embryo area vasculosa.

The chick embryo includes the area vasculosa is subdivided into 2 concentric zones, the inner transparent area pellucida vasculosa and the surrounding less transparent area opaca vasculosa, peripherally limited by the sinus terminalis. In this study, we have analyzed by a modern morphometric approach the total length of the vascular network, the number of vascular branches, of the branching points density, the modality of vessel ramification, and spatial arrangement of the vascular network in four consecutive stages of development of the area vasculosa. The results have shown that there is a significant 15% increase in the total length of the vascular network associated with a progressive increase of the number of vascular branches and of the branching points density. Moreover, the results indicated that vascular spatial disorder significantly decreased during development in area vasculosa, suggesting a more uniform occupancy of the tissue by the vascular pattern. Finally, a more regular pattern of branching was observed, as indicated by the significant decrease of topological disorder of the vascular tree.

Genetic induction and mechanochemical propagation of a morphogenetic wave.

Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. Here we report two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1-MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1-MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. We find that MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation.

Automated Macro Approach to Remove Vitelline Membrane Autofluorescence in Drosophila Embryo 4D Movies.

This chapter provides an ImageJ/Fiji automated macro approach to remove the vitelline membrane autofluorescence in live Drosophila embryo movies acquired in a 4D (3D plus time) fashion. The procedure consists in a segmentation pipeline that can cope with different relative intensities of the vitelline membrane autofluorescence, followed by a developed algorithm that adjusts the extracted outline selection to the shape deformations that naturally occur during Drosophila embryo development. Finally, the fitted selection is used to clear the external glowing halo that, otherwise, would obscure the visualization of the internal embryo labeling upon projection or 3D rendering.

Attachment of the blastoderm to the vitelline envelope affects gastrulation of insects.

During gastrulation, physical forces reshape the simple embryonic tissue to form the complex body plans of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the gastrulating tissue is surrounded by a rigid protective shell4. Although it is well-recognized that gastrulation movements depend on forces that are generated by tissue-intrinsic contractility5,6, it is not known whether interactions between the tissue and the protective shell provide additional forces that affect gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle (Tribolium castaneum) tightly adheres in a temporally coordinated manner to the vitelline envelope that surrounds the embryo. This attachment generates an additional force that counteracts tissue-intrinsic contractile forces to create asymmetric tissue movements. This localized attachment depends on an αPS2 integrin (inflated), and the knockdown of this integrin leads to a gastrulation phenotype that is consistent with complete loss of attachment. Furthermore, analysis of another integrin (the αPS3 integrin, scab) in the fruit fly (Drosophila melanogaster) suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline envelope. Our findings reveal a conserved mechanism through which the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable, counteracting forces that shape gastrulation movements in insects.

The role of metalloproteases in fertilisation in the ascidian Ciona robusta.

In the ascidian Ciona robusta (formerly C. intestinalis type A), the mechanism underlying sperm penetration through the egg investment remains unknown. We previously reported that proteins containing both an astacin metalloprotease domain and thrombospondin type 1 repeats are abundant in the sperm surface protein-enriched fraction of C. robusta. Here we investigated the involvement of those proteins in fertilisation. We refined the sequences of astacin metalloproteases, confirmed that five of them are present in the sperm, and labelled them as tunicate astacin and thrombospondin type 1 repeat-containing (Tast) proteins. Fertilisation of C. robusta eggs was potently inhibited by a metalloprotease inhibitor GM6001. The eggs cleaved normally when they were vitelline coat-free or the inhibitor was added after insemination. Furthermore, vitelline coat proteins were degraded after incubation with intact sperm. These results suggest that sperm metalloproteases are indispensable for fertilisation, probably owing to direct or indirect mediation of vitelline-coat digestion during sperm penetration. TALEN-mediated knockout of Tast genes and the presence of GM6001 impaired larval development at the metamorphic stage, suggesting that Tast gene products play a key role in late development.

Vitelline membrane proteins promote left-sided nodal expression after neurula rotation in the ascidian, Halocynthia roretzi.

Stereotyped left-right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the ascidian, Halocynthia roretzi. Neurula embryos rotate along the anterior-posterior axis in a counterclockwise direction, and the rotation stops when the left side of the embryo is oriented downwards, resulting in contact of the left-side epidermis with the vitelline membrane at the bottom of perivitelline space. Then, such contact induces the expression of nodal and its downstream Pitx2 gene in the left-side epidermis. Vitelline membrane is required for the promotion of nodal expression. Here, we showed that a chemical signal from the vitelline membrane promotes nodal gene expression, but mechanical stimulus at the point of contact is unnecessary since the treatment of devitellinated neurulae with an extract of the vitelline membrane promoted nodal expression on both sides. The signal molecules are already present in the vitelline membranes of unfertilized eggs. These signal molecules are proteins but not sugars. Specific fractions in gel filtration chromatography had the nodal promoting activity. By mass spectrometry, we selected 48 candidate proteins. Proteins that contain both a zona pellucida (ZP) domain and epidermal growth factor (EGF) repeats were enriched in the candidates of the nodal inducing molecules. Six of the ZP proteins had multiple EGF repeats that are only found in ascidian ZP proteins. These were considered to be the most viable candidates of the nodal-inducing molecules. Signal molecules are anchored to the entire vitelline membrane, and contact sites of signal-receiving cells are spatially and mechanically controlled by the neurula rotation. In this context, ascidians are unusual with respect to mechanisms for specification of the left-right axis. By suppressing formation of epidermis monocilia, we also showed that epidermal cilia drive the neurula rotation but are dispensable for sensing the signal from the vitelline membrane.