Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR-Cas9-mediated genome editing.

CRISPR-Cas9 genome editing has promising therapeutic potential for genetic diseases and cancers, but safety could be a concern. Here we use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing and in comparison to four parental cell lines. In addition to the previously reported large structural variants at on-target sites, we identify heretofore unexpected large chromosomal deletions (91.2 and 136 Kb) at atypical non-homologous off-target sites without sequence similarity to the sgRNA in two edited lines. The observed large structural variants induced by CRISPR-Cas9 editing in dividing cells may result in pathogenic consequences and thus limit the usefulness of the CRISPR-Cas9 editing system for disease modeling and gene therapy. In this work, our whole genomic analysis may provide a valuable strategy to ensure genome integrity after genomic editing to minimize the risk of unintended effects in research and clinical applications.

YULINK regulates vascular formation in zebrafish and HUVECs.

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs.

YULINK regulates vascular formation in zebrafish and HUVECs

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs. Key points Knockdown of YULINK with morpholino in embryos of double transgenic zebrafish exhibited abnormal venous formation. Tube formation and phosphorylated EPHB4 were decreased in YULINK knockdown HUVECs. FLIM-FRET, immunoprecipitation, as well as other imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B and TICAM2) and endosome markers (Clathrin and RHOB). Knockdown of YULINK decreased the internalization of VEGF and VEGFR2 in HUVECs.

Puf-A promotes cancer progression by interacting with nucleophosmin in nucleolus.

Previously, we identified Puf-A as a novel member of Puf-family RNA-binding proteins; however, its biological functions remain obscure. Analysis of tumor samples of non-small cell lung cancer (NSCLC) showed that high Puf-A expression correlated with high histology grade and abnormal p53 status. Kaplan-Meier curve for overall survival revealed high expression of Puf-A to predict poor prognosis in stage I NSCLC. Among patients with colorectal cancer, high Puf-A expression also showed an adverse impact on overall survival. In lung cancer cell lines, downregulation of p53 increased Puf-A expression, and upregulation of p53 dampened its expression. However, luciferase reporter assays indicated that PUF-A locus harbored the p53-response element, but regulated Puf-A transcription indirectly. In vivo suppression of p53 in CCSP-rtTA/TetO-Cre/LSL-KrasG12D/p53flox/flox conditional mutant mice accelerated the progression of the KrasG12D-driven lung cancer, along with enhanced expression of Puf-A. Importantly, intranasal delivery of shPuf-A to the inducible KrasG12D/p53flox/flox mice suppressed tumor progression. Puf-A silencing led to marked decreases in the 80S ribosomes, along with decrease in S6 and L5 in the cytoplasm and accumulation in the nucleolus. Based on immunofluorescence staining and immunoprecipitation studies, Puf-A interacted with NPM1 in nucleolus. Puf-A silencing resulted in NPM1 translocation from nucleolus to nucleoplasm and this disruption of NPM1 localization was reversed by a rescue experiment. Mechanistically, Puf-A silencing altered NPM1 localization, leading to the retention of ribosomal proteins in nucleolus and diminished ribosome biogenesis, followed by cell-cycle arrest/cell death. Puf-A is a potential theranostic target for cancer therapy and an important player in cancer progression.

Transmembrane and coiled-coil domain family 3 (TMCC3) regulates breast cancer stem cell and AKT activation.

Cancer stem cells (CSC) play a pivotal role in cancer metastasis and resistance to therapy. Previously, we compared the phosphoproteomes of breast cancer stem cells (BCSCs) enriched subpopulation and non-BCSCs sorted from breast cancer patient-derived xenograft (PDX), and identified a function unknown protein, transmembrane and coiled-coil domain family 3 (TMCC3) to be a potential enrichment marker for BCSCs. We demonstrated greater expression of TMCC3 in BCSCs than non-BCSCs and higher expression of TMCC3 in metastatic lymph nodes and lungs than in primary tumor of breast cancer PDXs. TMCC3 silencing suppressed mammosphere formation, ALDH activity and cell migration in vitro, along with reduced tumorigenicity and metastasis in vivo. Mechanistically, we found that AKT activation was reduced by TMCC3 silencing, but enhanced by TMCC3 overexpression. We further demonstrated that TMCC3 interacted directly with AKT through its 1-153 a.a. domain by cell-free biochemical assay in vitro and co-immunoprecipitation and interaction domain mapping assays in vivo. Based on domain truncation studies, we showed that the AKT-interacting domain of TMCC3 was essential for TMCC3-induced AKT activation, self-renewal, and metastasis. Clinically, TMCC3 mRNA expression in 202 breast cancer specimens as determined by qRT-PCR assay showed that higher TMCC3 expression correlated with poorer clinical outcome of breast cancer, including early-stage breast cancer. Multivariable analysis identified TMCC3 expression as an independent risk factor for survival. These findings suggest that TMCC3 is crucial for maintenance of BCSCs features through AKT regulation, and TMCC3 expression has independent prognostic significance in breast cancer. Thus, TMCC3 may serve as a new target for therapy directed against CSCs.

Yulink, predicted from evolutionary analysis, is involved in cardiac function.

BACKGROUND: The comparative evolutionary genomics analysis was used to study the functions of novel Ka/Ks-predicted human exons in a zebrafish model. The Yulink (MIOS, Entrez Gene: 54,468), a conserved gene from zebrafish to human with WD40 repeats at N-terminus, was identified and found to encode an 875 amino acid in human. The biological function of this Yulink gene in cardiomyocytes remains unexplored. The purpose of this study is to determine the involvement of Yulink in the functions of cardiomyocytes and to investigate its molecular regulatory mechanism. METHODS: Knockdown of Yulink was performed using morpholino or shRNA in zebrafish, mouse HL-1 cardiomyocytes, and human iPSC-derived cardiomyocytes. The expression levels of mRNA and protein were quantified by qPCR and western blots. Other methods including DNA binding, ligand uptake, agonists treatment and Ca2+ imaging assays were used to study the molecular regulatory mechanism by Yulink. Statistical data were shown as mean ± SD or mean ± standard error. RESULTS: The knockdown of yulink with three specific morpholinos in zebrafish resulted in cardiac dysfunctions with pericardial edema, decreased heart beats and cardiac output. The Yulink knockdown in mouse HL-1 cardiomyocytes disrupted Ca2+ cycling, reduced DNA binding activity of PPARγ (peroxisome proliferator-activated receptor gamma) and resulted in a reduction of Serca2 (sarcoplasmic reticulum Ca2+ ATPase 2) expression. Expression of Serca2 was up-regulated by PPARγ agonists and down-regulated by PPARγ-shRNA knockdown, suggesting that Yulink regulates SERCA2 expression through PPARγ in mouse HL-1 cardiomyocytes. On the other hand, YULINK, PPARγ or SERCA2 over-expression rescued the phenotypes of Yulink KD cells. In addition, knockdown of YULINK in human iPSC-derived cardiomyocytes also disrupted Ca2+ cycling via decreased SERCA2 expression. CONCLUSIONS: Overall, our data showed that Yulink is an evolutionarily conserved gene from zebrafish to human. Mechanistically Yulink regulated Serca2 expression in cardiomyocytes, presumably mediated through PPARγ nuclear entry. Deficiency of Yulink in mouse and human cardiomyocytes resulted in irregular Ca2+ cycling, which may contribute to arrhythmogenesis.

B3GALT5 knockout alters gycosphingolipid profile and facilitates transition to human naïve pluripotency.

Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of ß-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.

Modeling Alzheimer's Disease by Induced Pluripotent Stem Cells Carrying APP D678H Mutation.

Alzheimer's disease (AD), probably caused by abnormal accumulation of ß-amyloid (Aß) and aberrant phosphorylation of tau, is the most common cause of dementia among older people. Generation of patient-specific neurons by induced pluripotent stem cell (iPSC) technology facilitates exploration of the disease features in live human neurons from AD patients. In this study, we generated iPSCs from two familial AD patients carrying a heterozygous D678H mutation in the APP gene (AD-iPSCs). The neurons derived from our AD-iPSCs demonstrated aberrant accumulation of intracellular and secreted Aß42 and Aß40, reduction of serine 9 phosphorylation in glycogen synthase kinase 3ß (GSK3ß) hyperphosphorylation of threonine 181 and serine 396 in tau protein, impaired neurite outgrowth, downregulation of synaptophysin, and increased caspase 1 activity. The comparison between neurons derived from a sibling pair of wild-type and mutated iPSCs successfully recapitulated these AD phenotypes. Treatment with indole compound NC009-1 (3-((1H-Indole-3-yl)methyl)-4-(2-nitrophenyl)but-3-en-2-one), a potential Aß aggregation reducer, normalized the Aß levels and GSK3ß and tau phosphorylation, attenuated caspase 1 activity, and improved neurite outgrowth in AD-iPSC-derived neurons. Thus, APP D678H iPSCs-derived neurons recapitulate the cellular characteristics relevant to AD and enable exploration of the underlying pathogenesis and therapeutic strategies for AD.

RETRACTED: Deficiency of a novel gene, Yulink, predisposes to heart failure and ventricular arrhythmia.

Heart failure is a major cardiovascular disease and is associated with significant morbidity and mortality. Sudden cardiac death (SCD) in heart failure is a disastrous cardiovascular phenomenon. However, few studies have examined the genetic background that determines susceptibility to heart failure and SCD. We found that deficiency of a newly identified gene, Yulink, promoted cardiac alternans in zebrafish cardiomyocytes, and genetic knockdown (KD) resulted in pericardial edema, decreased cardiac output, and premature ventricular contractions. Yulink KD morphants exhibited irregular action potentials, slower Ca2+ reuptake, and alternans of Ca2+ transients and action potential duration, which are hallmarks for SCD susceptibility in heart failure. Similarly, KD of Yulink in mouse cardiomyocytes disrupted Ca2+ reuptake, reduced the expression of cardiac Serca2, and resulted in a reduction in peroxisome proliferator-activated receptor (PPAR)γ activity. Expression of Serca2 was up-regulated by PPARγ agonists and down-regulated by PPARγ-short hairpin RNA KD, suggesting that Yulink regulates Serca2 expression through PPARγ. Finally, Yulink and Serca2 were down-regulated in ventricular samples of hearts from patients with heart failure due to dilated cardiomyopathy. Our results highlight the interaction of Yulink with PPARγ in regulating Serca2 expression and suggest a mechanistic role of the Yulink in the development of human heart failure and SCD.-Tsai, C.-T., Kuo, M.-W., Lin, J.-L., Yu, A. L., Yu, J. Deficiency of a novel gene, Yulink, predisposes to heart failure and ventricular arrhythmia.

Hedgehog-PKA signaling and gnrh3 regulate the development of zebrafish gnrh3 neurons.

GnRH neurons secrete GnRH that controls the development of the reproduction system. Despite many studies, the signals controlling the development of GnRH neurons from its progenitors have not been fully established. To understand the development of GnRH neurons, we examined the development of gnrh3-expressing cells using a transgenic zebrafish line that expresses green fluorescent protein (GFP) and LacZ driven by the gnrh3 promoter. GFP and LacZ expression recapitulated that of gnrh3 in the olfactory region, olfactory bulb and telencephalon. Depletion of gnrh3 by morpholinos led to a reduction of GFP- and gnrh3-expressing cells, while over-expression of gnrh3 mRNA increased the number of these cells. This result indicates a positive feed-forward regulation of gnrh3 cells by gnrh3. The gnrh3 cells were absent in embryos that lack Hedgehog signaling, but their numbers were increased in embryos overexpressing shhb. We manipulated the amounts of kinase that antagonizes the Hedgehog signaling pathway, protein kinase A (PKA), by treating embryos with PKA activator forskolin or by injecting mRNAs encoding its constitutively active catalytic subunit (PKA*) and dominant negative regulatory subunit (PKI) into zebrafish embryos. PKA* misexpression or forskolin treatment decreased GFP cell numbers, while PKI misexpression led to ectopic production of GFP cells. Our data indicate that the Hedgehog-PKA pathway participates in the development of gnrh3-expressing neurons during embryogenesis.

A novel puf-A gene predicted from evolutionary analysis is involved in the development of eyes and primordial germ-cells.

Although the human genome project has been completed for some time, the issue of the number of transcribed genes with identifiable biological functions remains unresolved. We used zebrafish as a model organism to study the functions of Ka/Ks-predicted novel human exons, which were identified from a comparative evolutionary genomics analysis.In this study, a novel gene, designated as puf-A, was cloned and functionally characterized, and its homologs in zebrafish, mouse, and human were identified as one of the three homolog clusters which were consisted of 14 related proteins with Puf repeats. Computer modeling of human Puf-A structure and a pull-down assay for interactions with RNA targets predicted that it was a RNA-binding protein. Specifically, Puf-A contained a special six Puf-repeat domain, which constituted a unique superhelix half doughnut-shaped Puf domain with a topology similar to, but different from the conventional eight-repeat Pumilio domain. Puf-A transcripts were uniformly distributed in early embryos, but became restricted primarily to eyes and ovaries at a later stage of development. In mice, puf-A expression was detected primarily in retinal ganglion and pigmented cells. Knockdown of puf-A in zebrafish embryos resulted in microphthalmia, a small head, and abnormal primordial germ-cell (PGC) migration. The latter was confirmed by microinjecting into embryos puf-A siRNA containing nanos 3' UTR that expressed in PGC only. The importance of Puf-A in the maturation of germline stem cells was also implicated by its unique expression in the most primitive follicles (stage I) in adult ovaries, followed by a sharp decline of expression in later stages of folliculogenesis. Taken together, our study shows that puf-A plays an important role not only in eye development, but also in PGC migration and the specification of germ cell lineage. These studies represent an exemplary implementation of a unique platform to uncover unknown function(s) of human genes and their roles in development regulation.

A cyp19a1b-gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells.

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens.

c-Myb is an evolutionary conserved miR-150 target and miR-150/c-Myb interaction is important for embryonic development.

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.

Human TRIM71 and its nematode homologue are targets of let-7 microRNA and its zebrafish orthologue is essential for development.

Animal microRNAs (miRNAs) are short RNAs that function as posttranscriptional regulators of gene expression by binding to the target mRNAs. Noting that some miRNAs are highly conserved in evolution, we explored the possibility of evolutionary conservation of their targets. We identified human orthologues of experimentally verified let-7 miRNA target genes in Caenorhabditis elegans and used the luciferase reporter system to examine whether these human genes are still the targets of let-7 miRNA. We found that in some cases, the miRNA-target relationship has indeed been conserved in human. Interestingly, human TRIM71, an orthologue of C. elegans let-7-target lin-41 gene, can be repressed by hsa-let-7a and hsa-let-7c. This repression was abolished when both predicted let-7 target sites of TRIM71 were mutated. Moreover, the zebrafish lin-41 orthologue was also repressed by let-7 to a similar degree as was TRIM71. When the expression of zebrafish lin-41 orthologue was silenced by microinjection of RNA interference or morpholino into zebrafish zygotes, retarded embryonic development was observed, providing direct evidence for an essential role of lin-41 in zebrafish development. Taken together, our results suggest that the regulation of TRIM71 expression by let-7 has been evolutionarily conserved and that TRIM71 likely plays an important role in development.

Chromosomal organization, evolutionary relationship, and expression of zebrafish GnRH family members.

Multiple forms of gonadotropin-releasing hormone (GnRH) are found in different vertebrates. In this study, we have cloned cDNA encoding the full-length gnrh3 and gnrh2 from zebrafish brain and characterized their structure and expression patterns. We performed phylogenetic analysis and compared conserved syntenies in the region surrounding the GnRH genes from human, chicken, pufferfish, and zebrafish genores. The gnrh3 and gnrh2 genes were mapped to LG17 and LG21, respectively. The zebrafish genome appears to lack an ortholog to human GNRH1, and the human genome appears to lack an ortholog of gnrh3. Expression of gnrh3 began in the olfactory pit at 24-26 h postfertilization and expanded to the olfactory bulb during early larval stage. Expression of gnrh2 is always in the midbrain. In addition, GnRH is also expressed in boundary cells surrounding seminiferous cysts of the testis. Thus, this detailed phylogenetic, chromosomal comparison, and expression study defines the identity and the evolutionary relationship of two zebrafish gnrh genes. We propose a model describing the evolution of gnrh genes involving ancestral duplication of the genes followed by selective loss of one gene in some teleosts.

Gene duplication, gene loss and evolution of expression domains in the vertebrate nuclear receptor NR5A (Ftz-F1) family.

Fushi tarazu factor 1 (Ftz-F1, NR5A) is a zinc-finger transcription factor that belongs to the nuclear receptor superfamily and regulates genes that are involved in sterol and steroid metabolism in gonads, adrenals, liver and other tissues. To understand the evolutionary origins and developmental genetic relationships of the Ftz-F1 genes, we have cloned four homologous Ftz-f1 genes in zebrafish, called ff1a, ff1b, ff1c and ff1d. These four genes have different temporal and spatial expression patterns during development, indicating that they have distinct mechanisms of genetic regulation. Among them, the ff1a expression pattern is similar to mammalian Nr5a2, while the ff1b pattern is similar to that of mammalian Nr5a1. Genetic mapping experiments show that these four ff1 genes are located on chromosome segments conserved between the zebrafish and human genomes, indicating a common ancestral origin. Phylogenetic and conserved synteny analysis show that ff1a is the orthologue of NR5A2, and that ff1b and ff1d genes are co-orthologues of NR5A1 that arose by a gene-duplication event, probably a whole-genome duplication, in the ray-fin lineage, and each gene is located next to an NR6A1 co-orthologue as in humans, showing that the tandem duplication occurred before the divergence of human and zebrafish lineages. ff1c does not have a mammalian counterpart. Thus we have characterized the phylogenetic relationships, expression patterns and chromosomal locations of these Ftz-F1 genes, and have demonstrated their identities as NR5A genes in relation to the orthologous genes in other species.

Expression of zebrafish cyp11a1 as a maternal transcript and in yolk syncytial layer.

Cyp11a1 (P450scc, cholesterol side-chain cleavage enzyme) is the first enzyme for the synthesis of all steroid hormones. The regulation of steroid synthesis has been extensively investigated, except during embryogenesis. To study steroidogenesis in embryos, we have isolated the zebrafish cyp11a1 gene, which consists of 11 exons. Reverse transcription-polymerase chain reaction analysis indicates that zebrafish cyp11a1 is expressed temporally in two waves during embryonic stages and when sexual differentiation begins. It is expressed in adult brain, testicular Leydig cells, and the granulosa/theca layer of the ovary. In addition, zebrafish cyp11a1 is expressed in oocytes, and is inherited as a maternal transcript in early embryos. Throughout zebrafish epiboly and segmentation stages, cyp11a1 is expressed in the yolk syncytial layer. At 36 h post fertilization, cyp11a1 transcript is located ventral to the third somite, where the primordial interrenal gland is located. In summary, zebrafish cyp11a1 is expressed in the cytoplasm of oocytes, as a maternal transcript, and in yolk syncytial layer during early embryogenesis.