Sea Urchin Embryos Research Articles

Genome-wide analysis of chromatin accessibility using ATAC-seq.

Programs of gene transcription are controlled by cis-acting DNA elements, including enhancers, silencers, and promoters. Local accessibility of chromatin has proven to be a highly informative structural feature for identifying such regulatory elements, which tend to be relatively open due to their interactions with proteins. Recently, ATAC-seq (assay for transposase-accessible chromatin using sequencing) has emerged as one of the most powerful approaches for genome-wide chromatin accessibility profiling. This method assesses DNA accessibility using hyperactive Tn5 transposase, which simultaneously cuts DNA and inserts sequencing adaptors, preferentially in regions of open chromatin. ATAC-seq is a relatively simple procedure which can be applied to only a few thousand cells. It is well-suited to developing embryos of sea urchins and other echinoderms, which are a prominent experimental model for understanding the genomic control of animal development. In this chapter, we present a protocol for applying ATAC-seq to embryonic cells of sea urchins.

Methods for transplantation of sea urchin blastomeres.

The stereotypic cleavage pattern of sea urchin embryos provides a platform for dissection of early lineage decisions that lead to cell diversification. Cell transplantation provides a useful tool for understanding those decisions. The methods described in this paper provide a guide for how to produce embryonic mosaics in which either one cell is transplanted or an entire tier of cells are transplanted to a host embryo. Although the results of such a cut and paste experiment can be documented in many ways, one of the most useful approaches follows progeny of the transplanted cell as they go through morphogenesis using time-lapse imaging. Methods for mounting and imaging the embryos are provided.

In vivo analysis of protein translation activity in sea urchin eggs and embryos.

Protein synthesis is a major regulatory step of gene expression in different physiological processes including development. Translation of proteins in sea urchin is stimulated upon fertilization and is necessary for cell cycle progression and development. Translational control is exerted through multifactorial mechanisms, including mRNA recruitment into polysomes and increased rates of translational activity. In this chapter, we review the methods used in sea urchin eggs and embryos to analyze translation activity in vivo both from perspectives of the proteins and of the mRNAs. First, we describe methods to quantify or visualize newly synthesized proteins with radioactive and non-radioactive labeling techniques. Next we present the polysome isolation and profiling on sucrose gradients, allowing the identification of translated mRNAs. Finally, we outline a procedure to follow the translation of a reporter luciferase protein from an mRNA microinjected into the egg.

Methods for the experimental and computational analysis of gene regulatory networks in sea urchins.

The discovery of gene regulatory networks (GRNs) has opened a gate to access the genomic mechanisms controlling development. GRNs are systems of transcriptional regulatory circuits that control the differential specification of cell fates during development by regulating gene expression. The experimental analysis of GRNs involves a collection of methods, each revealing aspects of the overall control process. This review provides an overview of experimental and computational methods that have been successfully applied for solving developmental GRNs in the sea urchin embryo. The key in this approach is to obtain experimental evidence for functional interactions between transcription factors and regulatory DNA. In the second part of this review, a more generally applicable strategy is discussed that shows a path from experimental evidence to annotation of regulatory linkages to the generation of GRN models.

Analysis of microRNA functions.

MicroRNAs (miRNAs) are small non-coding RNAs that repress the translation and reduce the stability of target mRNAs in animal cells. Post-transcriptional regulation mediated by miRNAs is a highly conserved mechanism utilized by organisms throughout phylogeny to fine tune gene expression. We document the approaches used to study the function of a single miRNA and miRNA regulation of biological pathways in the sea urchin embryo. The protocols that are described include selection of miRNA inhibitors, test of miRNA direct targets, and the use of target protector morpholinos to evaluate the impact of miRNA inhibition on its targets. Using the described techniques and strategies, the sea urchin researcher will be able to validate a miRNA's direct targets and evaluate how inhibition of the miRNA affects developmental processes. These results will contribute to our understanding of the regulatory roles of miRNAs in development.

Comparative Toxicity Evaluation of Targeted Anticancer Therapeutics in Embryonic Zebrafish and Sea Urchin Models

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can pro-mote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhib-ited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORCl/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy.

CRISPR/Cas9-mediated genome editing in sea urchins.

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) technology enables rapid, targeted, and efficient changes in the genomes of various model organisms. The short guide RNAs (gRNAs) of the CRISPR/Cas9 system can be designed to recognize target DNA within coding regions for functional gene knockouts. Several studies have demonstrated that the CRISPR/Cas9 system efficiently and specifically targets sea urchin genes and results in expected mutant phenotypes. In addition to disrupting gene functions, modifications and additions to the Cas9 protein enable alternative activities targeted to specific sites within the genome. This includes a fusion of cytidine deaminase to Cas9 (Cas9-DA) for single nucleotide conversion in targeted sites. In this chapter, we describe detailed methods for the CRISPR/Cas9 application in sea urchin embryos, including gRNA design, in vitro synthesis of single guide RNA (sgRNA), and the usages of the CRISPR/Cas9 technology for gene knockout and single nucleotide editing. Methods for genotyping the resultant embryos are also provided for assessing efficiencies of gene editing.

Neurogenesis in the sea urchin embryo is initiated uniquely in three domains.

Many marine larvae begin feeding within a day of fertilization, thus requiring rapid development of a nervous system to coordinate feeding activities. Here, we examine the patterning and specification of early neurogenesis in sea urchin embryos. Lineage analysis indicates that neurons arise locally in three regions of the embryo. Perturbation analyses showed that when patterning is disrupted, neurogenesis in the three regions is differentially affected, indicating distinct patterning requirements for each neural domain. Six transcription factors that function during proneural specification were identified and studied in detail. Perturbations of these proneural transcription factors showed that specification occurs differently in each neural domain prior to the Delta-Notch restriction signal. Though gene regulatory network state changes beyond the proneural restriction are largely unresolved, the data here show that the three neural regions already differ from each other significantly early in specification. Future studies that define the larval nervous system in the sea urchin must therefore separately characterize the three populations of neurons that enable the larva to feed, to navigate, and to move food particles through the gut.

A vesicular Na+/Ca2+ exchanger in coral calcifying cells.

The calcium carbonate skeletons of corals provide the underlying structure of coral reefs; however, the cellular mechanisms responsible for coral calcification remain poorly understood. In osteoblasts from vertebrate animals, a Na+/Ca2+ exchanger (NCX) present in the plasma membrane transports Ca2+ to the site of bone formation. The aims of this study were to establish whether NCX exists in corals and its localization within coral cells, which are essential first steps to investigate its potential involvement in calcification. Data mining identified genes encoding for NCX proteins in multiple coral species, a subset of which were more closely related to NCXs from vertebrates (NCXA). We cloned NCXA from Acropora yongei (AyNCXA), which, unexpectedly, contained a peptide signal that targets proteins to vesicles from the secretory pathway. AyNCXA subcellular localization was confirmed by heterologous expression of fluorescently tagged AyNCXA protein in sea urchin embryos, which localized together with known markers of intracellular vesicles. Finally, immunolabeling of coral tissues with specific antibodies revealed AyNCXA was present throughout coral tissue. AyNCXA was especially abundant in calcifying cells, where it exhibited a subcellular localization pattern consistent with intracellular vesicles. Altogether, our results demonstrate AyNCXA is present in vesicles in coral calcifying cells, where potential functions include intracellular Ca2+ homeostasis and Ca2+ transport to the growing skeleton as part of an intracellular calcification mechanism.

Canonical and non-canonical Wnt signaling pathways define the expression domains of Frizzled 5/8 and Frizzled 1/2/7 along the early anterior-posterior axis in sea urchin embryos

The spatiotemporal expression of Frizzled receptors is critical for patterning along the early anterior-posterior axis during embryonic development in many animal species. However, the molecular mechanisms that regulate the expression of Frizzled receptors are incompletely understood in any species. In this study, I examine how the expression of two Frizzled receptors, Fzl1/2/7 and Fzl5/8, is controlled by the Wnt signaling network which directs specification and positioning of early regulatory states along the anterior-posterior (AP) axis of sea urchin embryos. I used a combination of morpholino- and dominant negative-mediated interference to knock down each Wnt signaling pathway involved in the AP Wnt signaling network. I found that the expression of zygotic fzl5/8 as well as that of the anterior neuroectoderm gene regulatory network (ANE GRN) is activated by an unknown broadly expressed regulatory state and that posterior Wnt/β-catenin signaling is necessary to down regulate fzl5/8's expression in posterior blastomeres. I show that zygotic expression of fzl1/2/7 in the equatorial ectodermal belt is dependent on an uncharacterized regulatory mechanism that works in the same cells receiving the TGF-β signals patterning this territory along the dorsal-ventral axis. In addition, my data indicate that Fzl1/2/7 signaling represses its own expression in a negative feedback mechanism. Finally, we discovered that a balance between the activities of posterior Wnt8 and anterior Dkk1 is necessary to establish the correct spatial expression of zygotic fzl12/7 expression in the equatorial ectodermal domain during blastula and gastrula stages. Together, these studies lead to a better understanding of the complex interactions among the three Wnt signaling pathway governing AP axis specification and patterning in sea urchin embryos.

MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo.

In the sea urchin embryo, specification of the dorsal-ventral axis critically relies on the spatially restricted expression of nodal in the presumptive ventral ectoderm. The ventral restriction of nodal expression requires the activity of the maternal TGF-β ligand Panda but the mechanism by which Panda restricts nodal expression is unknown. Similarly, what initiates expression of nodal in the ectoderm and what are the mechanisms that link patterning along the primary and secondary axes is not well understood. We report that in Paracentrotus lividus, the activity of the maternally expressed ETS-domain transcription factor Yan/Tel is essential for the spatial restriction of nodal. Inhibiting translation of maternal yan/tel mRNA disrupted dorsal-ventral patterning in all germ layers by causing a massive ectopic expression of nodal starting from cleavage stages, mimicking the phenotype caused by inactivation of the maternal Nodal antagonist Panda. We show that like in the fly or in vertebrates, the activity of sea urchin Yan/Tel is regulated by phosphorylation by MAP kinases. However, unlike in the fly or in vertebrates, phosphorylation by GSK3 plays a central role in the regulation Yan/Tel stability in the sea urchin. We show that GSK3 phosphorylates Yan/Tel in vitro at two different sites including a β-TRCP ubiquitin ligase degradation motif and a C-terminal Ser/Thr rich cluster and that phosphorylation of Yan/Tel by GSK3 triggers its degradation by a β-TRCP/proteasome pathway. Finally, we show that, Yan is epistatic to Panda and that the activity of Yan/Tel is required downstream of Panda to restrict nodal expression. Our results identify Yan/Tel as a central regulator of the spatial expression of nodal in Paracentrotus lividus and uncover a key interaction between the gene regulatory networks responsible for patterning the embryo along the dorsal-ventral and animal-vegetal axes.

The impact of propranolol, 17α-ethinylestradiol, and gemfibrozil on early life stages of marine organisms: effects and risk assessment.

Pharmaceuticals are ubiquitously detected in the marine environment at the ng-μg/L range. Given their biological activity, these compounds are known to induce detrimental effects on biota at relatively low exposure levels; however, whether they affect early life stages of marine species is still unclear. In this study, a set of bioassays was performed to assess the effects of propranolol (PROP), 17-α ethinylestradiol (EE2), and gemfibrozil (GEM) on gamete fertilization and embryonic development of mussels (Mytilus galloprovincialis) and sea urchins (Paracentrotus lividus), and on the survival of seabream (Sparus aurata) larvae. Treatments of PROP (500, 5000, 50,000ng/L), EE2 (5, 50, 500ng/L), and GEM (50, 500, 5000ng/L) were selected to encompass levels comparable or superior to environmental concentrations. Obtained data were tested for dose-response curve fitting and the lowest EC10/LC10 used to calculate risk quotients (RQs) based on the MEC/PNEC. No alteration was induced by PROP on the mussel gamete fertilization, while inhibitory effects were observed at environmental levels of EE2 (500ng/L) and GEM (5000ng/L). Fertilization was significantly reduced in sea urchin at all PROP and EE2 dosages. The 48-h exposure to all pharmaceuticals induced the onset of morphological abnormalities in either mussel or sea urchin embryos. Alterations were generally observed at environmentally relevant dosages, except for PROP in mussels, in which alterations occurred only at 50,000ng/L. A decreased survival of seabream larvae was recorded after 96-h exposure to PROP (all treatments), EE2 (50-500ng/L), and GEM (500ng/L). A median RQ > 1 was obtained for all pharmaceuticals, assigning a high risk to their occurrence in marine environments. Overall, results showed that current levels of contamination by pharmaceuticals can impact early stages of marine species, which represent critical junctures in the resilience of coastal ecosystems.

Development of the sea urchin Heliocidaris crassispina from Hong Kong is robust to ocean acidification and copper contamination

Metallic pollution is of particular concern in coastal cities. In the Asian megacity of Hong Kong, despite water qualities have improved over the past decade, some local zones are still particularly affected and could represent sinks for remobilization of labile toxic species such as copper. Ocean acidification is expected to increase the fraction of the most toxic form of copper (Cu2+) by 2.3-folds by 2100 (pH ≈7.7), increasing its bioavailability to marine organisms. Additionally, multiple stressors are likely to exert concomitant effects (additive, synergic or antagonist) on the organisms living in the sea.Here, we tested the hypothesis that copper-contaminated waters are more toxic to sea urchin larvae under future pH conditions. We exposed sea urchin embryos and larvae to two low-pH and two copper treatments (0.1 and 1.0 μM) in three separate experiments. Over the short time typically used for toxicity tests (up to 4-arm plutei, i.e. 3 days), larvae of the sea urchin Heliocidaris crassispina were robust and survived the copper levels present in Hong Kong waters today (≤0.19 μM) as well as the average pH projected for 2100. We, however, observed significant mortality with lowering pH in the longer, single-stressor experiment (Expt A: 8-arm plutei, i.e. 9 days). Abnormality and arm asymmetry were significantly increased by pH or/and by copper presence (depending on the experiment and copper level). Body size (d3; but not body growth rates in Expt A) was significantly reduced by both lowered pH and added copper. Larval respiration (Expt A) was doubled by a decrease at pHT from 8.0 to 7.3 on d6. In Expt B1.0 and B0.1, larval morphology (relative arm lengths and stomach volume) were affected by at least one of the two investigated factors.Although the larvae appeared robust, these sub-lethal effects may have indirect consequences on feeding, swimming and ultimately survival. The complex relationship between pH and metal speciation/uptake is not well-characterized and further investigations are urgently needed to detangle the mechanisms involved and to identify possible caveats in routinely used toxicity tests.

Using rapid assessment of marine pollution (RAMP) techniques to assess the quality of marine sediments

Sediments represent an important environmental compartment, because they provide substrate for a range of species and may accumulate contaminants in high concentrations. However, the universe of methods to assess the quality of sediments is still small. This investigation aimed to assess the quality of sediments from some sites of Ubatuba (SE Brazil). To achieve that, sediments were analyzed for the presence of PAHs, by immunoassay ELISA kit for the carcinogenic PAH (c-PAH) RaPID Assay, and for toxicity to amphipods, sea-urchin embryos and direct exposure of mussel hemocytes and measurements of the nuetral red retention time (NRTT). Results showed higher levels of PAHs in sediments from Itagua and Ribeira, those more intensely affected by contamination sources. The ecotoxicological analyses indicated these two sites as more degraded, together with the sediments from Lamberto Beach, which is also influenced by nautical activities. The NRRT correlated with the quantities of PAHs in sediments. We concluded that Ribeira Bay and Itagua Beach need more detailed investigation on pollution and that the NRRT assay exposing directly hemocytes to sediment elutriates can be a useful tool to assessing sediment quality.

Extremely stable high molecular mass soluble multiprotein complex from eggs of sea urchin Strongylocentrotus intermedius with phosphatase activity.

It was proposed that most biological processes are performed by different protein complexes. In contrast to individual proteins and enzymes, their complexes usually have other biological functions, and their formation may be important system process for the expansion of diversity and biological functions of different molecules. Identification and characterization of embryonic components including proteins and their multiprotein complexes seem to be very important for an understanding of embryo function. We have isolated and analyzed for the first time a very stable multiprotein complex (SPC; approximately 1100kDa) from the soluble fraction of extracts of the sea urchin embryos. By fast protein liquid chromatography (FPLC) gel filtration the SPC was well separated from other extract proteins. Stable multiprotein complex is stable in different drastic conditions but dissociates moderately in the presence of 8M urea+1.0M NaCl. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis data, this complex contains many major, moderate and minor proteins with molecular masses from 10 to 95kDa. The SPC was destroyed by 8M urea or SDS, and its components were separated using thin layer chromatography, ion-exchange chromatography, gel filtration, and reverse phase chromatography. Using matrix-assisted laser desorption/ionization mass spectrometry of partially dissociated SPC, it was shown that the complex contains not only proteins (10-95kDa) but also few dozens of peptides with molecular masses from 2 to 9.5kDa. Short peptides form very strong complexes, which at the treatment of SPC with urea or SDS can be partially break down into smaller complexes having different peptide compositions. Reverse phase chromatography of these complexes after all type of abovementioned chromatographies led to detection from 6 to 11 distinct peaks corresponding to new complexes containing up to a few dozens of peptides. The SPCs possess alkaline phosphatase activity. Progress in the study of embryos protein complexes can help to understand their biological functions.

Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava

BackgroundMesoderm is generally considered to be a germ layer that is unique to Bilateria, and it develops into diverse tissues, including muscle, and in the case of vertebrates, the skeleton and notochord. Studies on various deuterostome animals have demonstrated that fibroblast growth factor (FGF) signaling is required for the formation of many mesodermal structures, such as vertebrate somites, from which muscles are differentiated, and muscles in sea urchin embryos, suggesting an ancient role of FGF signaling in muscle development. However, the formation of trunk muscles in invertebrate chordates is FGF-independent, leading to ambiguity about this ancient role in deuterostomes. To further understand the role of FGF signaling during deuterostome evolution, we investigated the development of mesodermal structures during embryogenesis and metamorphosis in Ptychodera flava, an indirect-developing hemichordate that has larval morphology similar to echinoderms and adult body features that are similar to chordates.ResultsHere we show that genes encoding FGF ligands, FGF receptors and transcription factors that are known to be involved in mesoderm formation and myogenesis are expressed dynamically during embryogenesis and metamorphosis. FGF signaling at the early gastrula stage is required for the specification of the mesodermal cell fate in P. flava. The mesoderm cells are then differentiated stepwise into the hydroporic canal, the pharyngeal muscle and the muscle string; formation of the last two muscular structures are controlled by FGF signaling. Moreover, augmentation of FGF signaling during metamorphosis accelerated the process, facilitating the transformation from cilia-driven swimming larvae into muscle-driven worm-like juveniles.ConclusionsOur data show that FGF signaling is required for mesoderm induction and myogenesis in the P. flava embryo, and it is reiteratively used for the morphological transition during metamorphosis. The dependence of muscle development on FGF signaling in both planktonic larvae and sand-burrowing worms supports its ancestral role in deuterostomes.

Release of Sticky Glycoproteins from Chlamydomonas Flagella During Microsphere Translocation on the Surface Membrane.

Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.

Efficient hydrogenation of benzaldoximes and Schiff bases on ceramic high-porosity palladium catalysts

An efficient catalytic method for the synthesis of benzyl- and dibenzylamines by hydrogenating oximes and Schiffbases was developed on palladium supported high-porosity foamed ceramic block catalyst. The multiple regeneration ability of the foamed ceramic block catalyst can significantly decrease the Pd consumption as compared to the use of the conventional 10%Pd/C catalyst. Owing to a high hardness of the foamed ceramic catalyst, the reaction mixture can rapidly be removed from the reactor without using filtering devices. The structures produced by the reaction are fragments of biologically active and natural molecules. Antiproliferative properties of dibenzylamines revealed on the sea urchin embryo model suggest that these compounds can be considered as promising agents for the design of new anticancer drugs.

Perturbation of Developmental Regulatory Gene Expression by a G-Quadruplex DNA Inducer in the Sea Urchin Embryo.

The G-quadruplex (G4) is a four-stranded DNA structure identified in vivo in guanine-rich regions located in the promoter of a number of genes. Intriguing evidence suggested that small molecules acting as G4-targeting ligands could potentially regulate multiple cellular processes via either stabilizing or disruptive effects on G4 motifs. Research in this field aims to prove the direct role of G4 ligands and/or structures on a specific biological process in a complex living organism. In this study, we evaluate in vivo the effects of a nickel(II)-salnaphen-like complex, named Nisaln, a potent G4 binder and stabilizer, during embryogenesis of the sea urchin embryo. We describe developmental defects inflicted by Nisaln and correlate them with variation in the expression of several regulatory genes. It is worth mentioning that we show that Nisaln binds a G4 structure in the promoter of hbox12-a, a gene lying at the top of the developmental regulatory hierarchy, inducing overexpression of this gene.

An optogenetic approach to control protein localization during embryogenesis of the sea urchin

Light inducible protein–protein interactions have been used to manipulate protein localization and function in the cell with utmost spatial and temporal precision. In this technical report, we use a recently developed optogenetic approach to manipulate protein localization in the developing sea urchin embryo. A photosensitive LOV domain from Avena sativa phototropin1 cages a small peptide that binds the engineered PDZ domain (ePDZ) upon blue light irradiation. Using this system, mCherry tagged proteins fused with the LOV domain were recruited to ectopic sub-cellular regions such as the membrane, microtubules, or actin by GFP tagged proteins fused with the ePDZ domain upon blue light irradiation within 1–3 min in the sea urchin embryo. The efficiency and speed of recruitment of each protein to its respective subcellular region appeared to be dependent on the power and duration of laser irradiation, as well as the respective level of affinity to the tagged location. Controlled laser irradiation allowed partial recruitment of the spindle to the membrane, and resulted in cell blebbing. Vasa, a cell cycle and germline factor that localizes on the spindle and enriches in the micromeres at 8–16 cell stage was recruited to ectopic sites, preventing normal enrichment. Continuous blue light activation with a regular blue aquarium light over two days of culture successfully induced LOV-ePDZ binding in the developing embryos, resulting in continued ectopic recruitment of Vasa and failure in gastrulation at Day 2. Although some cytotoxicity was observed with prolonged blue light irradiation, this optogenetic system provides a promising approach to test the sub-cellular activities of developmental factors, as well as to alter protein localization and development during embryogenesis.