Visualizing metabolic regulation using metabolic biosensors during sea urchin embryogenesis.

Growing evidence suggests that metabolic regulation directly influences cellular function and development and thus may be more dynamic than previously expected. In vivo and in real-time analysis of metabolite activities during development is crucial to test this idea directly. In this study, we employ two metabolic biosensors to track the dynamics of pyruvate and oxidative phosphorylation (Oxphos) during the early embryogenesis of the sea urchin. A pyruvate sensor, PyronicSF, shows the signal enrichment on the mitotic apparatus, which is consistent with the localization patterns of the corresponding enzyme, pyruvate kinase (PKM). The addition of pyruvate increases the PyronicSF signal, while PKM knockdown decreases its signal, responding to the pyruvate level in the cell. Similarly, a ratio-metric sensor, Grx-roGFP, that reads the redox potential of the cell responds to DTT and H2O2, the known reducer and inducer of Oxphos. These observations suggest that these metabolic biosensors faithfully reflect the metabolic status in the cell during embryogenesis. The time-lapse imaging of these biosensors suggests that pyruvate and Oxphos levels change both spatially and temporarily during embryonic development. Pyruvate level is increased first in micromeres compared to other blastomeres at the 16-cell stage and remains high in ectoderm while decreasing in endomesoderm during gastrulation. In contrast, the Oxphos signal first decreases in micromeres at the 16-cell stage, while it increases in the endomesoderm during gastrulation, showing the opposite trend of the pyruvate signal. These results suggest that metabolic regulation is indeed both temporally and spatially dynamic during embryogenesis, and these biosensors are a valuable tool to monitor metabolic activities in real-time in developing embryos.

The evolutionary modifications of a GoLoco motif in the AGS protein facilitate micromere formation in the sea urchin embryo.

The evolutionary introduction of asymmetric cell division (ACD) into the developmental program facilitates the formation of a new cell type, contributing to developmental diversity and, eventually, to species diversification. The micromere of the sea urchin embryo may serve as one of those examples: An ACD at the 16-cell stage forms micromeres unique to echinoids among echinoderms. We previously reported that a polarity factor, Activator of G-protein Signaling (AGS), plays a crucial role in micromere formation. However, AGS and its associated ACD factors are present in all echinoderms and across most metazoans, leaving a question of what evolutionary modification of AGS protein or its surrounding molecular environment contributed to the evolutionary acquisition of micromeres only in echinoids. In this study, we learned that the GoLoco motifs at the AGS C-terminus play critical roles in regulating micromere formation in sea urchin embryos. Further, other echinoderms' AGS or chimeric AGS that contain the C-terminus of AGS orthologs from various organisms showed varied localization and function in micromere formation. In contrast, the sea star or the pencil urchin orthologs of other ACD factors were consistently localized at the vegetal cortex in the sea urchin embryo, suggesting that AGS may be a unique variable factor that facilitates ACD diversity among echinoderms. Consistently, sea urchin AGS appears to facilitate micromere-like cell formation and accelerate the enrichment timing of the germline factor Vasa during early embryogenesis of the pencil urchin, an ancestral type of sea urchin. Based on these observations, we propose that the molecular evolution of a single polarity factor facilitates ACD diversity while preserving the core ACD machinery among echinoderms and beyond during evolution.

Characterization of p53/p63/p73 and Myc expressions during embryogenesis of the sea urchin.

BACKGROUND: Some marine invertebrate organisms are considered not to develop tumors due to unknown mechanisms. To gain an initial insight into how tumor-related genes may be expressed and function during marine invertebrate development, we here leverage sea urchin embryos as a model system and characterize the expressions of Myc and p53/p63/p73 which are reported to function synergistically in mammalian models as an oncogene and tumor suppressor, respectively. RESULTS: During sea urchin embryogenesis, a combo gene of p53/p63/p73 is found to be maternally loaded and decrease after fertilization both in transcript and protein, while Myc transcript and protein are zygotically expressed. p53/p63/p73 and Myc proteins are observed in the cytoplasm and nucleus of every blastomere, respectively, throughout embryogenesis. Both p53/p63/p73 and Myc overexpression results in compromised development with increased DNA damage after the blastula stage. p53/p63/p73 increases the expression of parp1, a DNA repair/cell death marker gene, and suppresses endomesoderm gene expressions. In contrast, Myc does not alter the expression of specification genes or oncogenes yet induces disorganized morphology. CONCLUSIONS: p53/p63/p73 appears to be important for controlling cell differentiation, while Myc induces disorganized morphology yet not through conventional oncogene regulations or apoptotic pathways during embryogenesis of the sea urchin.

Vasa, a regulator of localized mRNA translation on the spindle.

Localized mRNA translation is a biological process that allows mRNA to be translated on-site, which is proposed to provide fine control in protein regulation, both spatially and temporally within a cell. We recently reported that Vasa, an RNA-helicase, is a promising factor that appears to regulate this process on the spindle during the embryonic development of the sea urchin, yet the detailed roles and functional mechanisms of Vasa in this process are still largely unknown. In this review article, to elucidate these remaining questions, we first summarize the prior knowledge and our recent findings in the area of Vasa research and further discuss how Vasa may function in localized mRNA translation, contributing to efficient protein regulation during rapid embryogenesis and cancer cell regulation.

The germline factor DDX4 contributes to the chemoresistance of small cell lung cancer cells.

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DEAD-box helicase 4 (DDX4), a germline factor and RNA helicase conserved in all multicellular organisms, contributes to increased cell motility and cisplatin-mediated drug resistance in small cell lung cancer (SCLC) cells. Proteomic analysis suggests that DDX4 expression upregulates proteins related to DNA repair and immune/inflammatory response. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Further, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of immune/inflammatory response markers. Taken together, we propose that DDX4 increases SCLC cell survival, by increasing the DNA damage and immune response pathways, especially under challenging conditions such as cisplatin treatment.

MPS1 inhibition primes immunogenicity of KRAS-LKB1 mutant lung cancer.

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in T cell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores T cell infiltration in vivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.

Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions.

mRNA translation on the spindle is hypothesized to be an essential strategy for the localized production of cell regulators. This mechanism may be important particularly in early embryonic cells, which have a large diffusion volume and that undergo rapid cell divisions. Evidence to test such a hypothesis has been, however, limited. Here, we use an embryo with both symmetric and asymmetric cell divisions and manipulate Vasa protein, an RNA-helicase, on the spindle in live sea urchin embryos. We learned that the spindle serves as a major site of translation and that protein synthesis within a single spindle can be unequal and help drive asymmetric cell divisions during embryogenesis. Recruiting Vasa to the ectopic sub-cellular region induced a new site of translation, disturbed asymmetric translation on the spindle, and changed the cell fate. Based on these observations, we conclude that Vasa functions in localized translation, which provides a spatiotemporal control in protein synthesis and is essential for rapidly developing embryonic cells.

Micromere formation and its evolutionary implications in the sea urchin.

The micromeres of the sea urchin embryo are distinct from other blastomeres. After they arise through an asymmetric cell division at the 8- to 16-cell stage, micromeres immediately function as organizers. They also commit themselves to specific cell fates such as larval skeletogenic cells and primordial germ cells, while other blastomeres remain plastic and uncommitted at the 16-cell stage. In the phylum Echinodermata, only the sea urchin (class Echinoidea) embryo forms micromeres that serve as apparent organizers during early embryogenesis. Therefore, it is considered that micromeres are the derived features and that modification(s) of the developmental system allowed evolutionary introduction of this unique cell lineage. In this chapter, we summarize the both historic and recent observations that demonstrate unique properties of micromeres and discuss how this lineage of micromeres may have arisen during echinoderm evolution.

Sperm lacking Bindin are infertile but are otherwise indistinguishable from wildtype sperm.

Cell-cell fusion is limited to only a few cell types in the body of most organisms and sperm and eggs are paradigmatic in this process. The specialized cellular mechanism of fertilization includes the timely exposure of gamete-specific interaction proteins by the sperm as it approaches the egg. Bindin in sea urchin sperm is one such gamete interaction protein and it enables species-specific interaction with a homotypic egg. We recently showed that Bindin is essential for fertilization by use of Cas9 targeted gene inactivation in the sea urchin, Hemicentrotus pulcherrimus. Here we show phenotypic details of Bindin-minus sperm. Sperm lacking Bindin do not bind to nor fertilize eggs at even high concentrations, yet they otherwise have wildtype morphology and function. These features include head shape, tail length and beating frequency, an acrosomal vesicle, a nuclear fossa, and they undergo an acrosomal reaction. The only phenotypic differences between wildtype and Bindin-minus sperm identified is that Bindin-minus sperm have a slightly shorter head, likely as a result of an acrosome lacking Bindin. These data, and the observation that Bindin-minus embryos develop normally and metamorphose into normal functioning adults, support the contention that Bindin functions are limited to species-specific sperm-egg interactions. We conclude that the evolutionary divergence of Bindin is not constrained by any other biological roles.

Bindin is essential for fertilization in the sea urchin.

Species-specific sperm-egg interactions are essential for sexual reproduction. Broadcast spawning of marine organisms is under particularly stringent conditions, since eggs released into the water column can be exposed to multiple different sperm. Bindin isolated from the sperm acrosome results in insoluble particles that cause homospecific eggs to aggregate, whereas no aggregation occurs with heterospecific eggs. Therefore, Bindin is concluded to play a critical role in fertilization, yet its function has never been tested. Here we report that Cas9-mediated inactivation of the bindin gene in a sea urchin results in perfectly normal-looking embryos, larvae, adults, and gametes in both males and females. What differed between the genotypes was that the bindin-/- sperm never fertilized an egg, functionally validating Bindin as an essential gamete interaction protein at the level of sperm-egg cell surface binding.

Light-induced, spatiotemporal control of protein in the developing embryo of the sea urchin.

Differential protein regulation is a critical biological process that regulates cellular activity and controls cell fate determination. It is especially important during early embryogenesis when post-transcriptional events predominate differential fate specification in many organisms. Light-induced approaches have been a powerful technology to interrogate protein functions with temporal and spatial precision, even at subcellular levels within a cell by controlling laser irradiation on the confocal microscope. However, application and efficacy of these tools need to be tested for each model system or for the cell type of interest because of the complex nature of each system. Here, we introduce two types of light-induced approaches to track and control proteins at a subcellular level in the developing embryo of the sea urchin. We found that the photoconvertible fluorescent protein Kaede is highly efficient to distinguish pre-existing and newly synthesized proteins with no apparent phototoxicity, even when interrogating proteins associated with the mitotic spindle. Further, chromophore-assisted light inactivation (CALI) using miniSOG successfully inactivated target proteins of interest in the vegetal cortex and selectively delayed or inhibited asymmetric cell division. Overall, these light-induced manipulations serve as important molecular tools to identify protein function for for subcellular interrogations in developing embryos.

Functional contribution of DCLKs in sea urchin development.

BACKGROUND: Doublecortin-like kinase1 and 2 (DCLKs) are protein Ser/Thr kinases important for neuronal development. More recently, they are also reported to regulate plasticity such as cell proliferation and differentiation of stem cells and cancer cells, but the details of their functions in this biological context are still unclear. With an attempt to reveal the functions of DCLKs in plasticity regulation, we here used the sea urchin embryo that undergoes highly regulative development as an experimental model. RESULTS: We found that both the transcripts and the proteins of DCLKs are uniformly present during early embryogenesis and with some enrichment in mesenchymal cells after gastrula stage. Knockdown of DCLKs induced general developmental delay and defects at day 2. Further, the damage on the embryo/larva induced ectopic expression of DCLKs in the ectoderm where the damage was most severe. Under a tumor-prone or -suppressive condition, DCLKs expression was upregulated or downregulated, respectively, after damage. In both cases, the embryos showed severe developmental defects. CONCLUSIONS: Taken together, a transient upregulation of DCLKs appears to be involved in a damage response both during normal and abnormal development, and which could result in different phenotypes in a context dependent manner.

CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin.

We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-ß signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.

Localized translation on the mitotic apparatus: A history and perspective.

Localized translation is a proposed biological event that allows mRNA to be translated on site, providing an additional level of protein regulation within a cell. Examples of localized translation have been found or proposed in a variety of cellular contexts from neurons to cancer cells and implicated in both normal development and disease for over a half century. For example, mRNA translation on the mitotic apparatus (MA) was initially hypothesized in the 1950-60s. However, its proof of existence, biological significance and mechanistic details have remained sparse and it is still unclear how well conserved this mechanism may be among different cell types or organisms. In this review, we provide a brief historic summary of translation on the MA and discuss how current and future work may help us understand this biological process that provides a subcellular level of regulation in protein synthesis within a cell.

Diversity of activator of G-protein signaling (AGS)-family proteins and their impact on asymmetric cell division across taxa.

Asymmetric cell division (ACD) is a cellular process that forms two different cell types through a cell division and is thus critical for the development of all multicellular organisms. Not all but many of the ACD processes are mediated by proper orientation of the mitotic spindle, which segregates the fate determinants asymmetrically into daughter cells. In many cell types, the evolutionarily conserved protein complex of Gαi/AGS-family protein/NuMA-like protein appears to play critical roles in orienting the spindle and/or generating the polarized cortical forces to regulate ACD. Studies in various organisms reveal that this conserved protein complex is slightly modified in each phylum or even within species. In particular, AGS-family proteins appear to be modified with a variable number of motifs in their functional domains across taxa. This apparently creates different molecular interactions and mechanisms of ACD in each developmental program, ultimately contributing to developmental diversity across species. In this review, we discuss how a conserved ACD machinery has been modified in each phylum over the course of evolution with a major focus on the molecular evolution of AGS-family proteins and its impact on ACD regulation.

Genetic manipulation of the pigment pathway in a sea urchin reveals distinct lineage commitment prior to metamorphosis in the bilateral to radial body plan transition.

Echinoderms display a vast array of pigmentation and patterning in larval and adult life stages. This coloration is thought to be important for immune defense and camouflage. However, neither the cellular nor molecular mechanism that regulates this complex coloration in the adult is known. Here we knocked out three different genes thought to be involved in the pigmentation pathway(s) of larvae and grew the embryos to adulthood. The genes tested were polyketide synthase (PKS), Flavin-dependent monooxygenase family 3 (FMO3) and glial cells missing (GCM). We found that disabling of the PKS gene at fertilization resulted in albinism throughout all life stages and throughout all cells and tissues of this animal, including the immune cells of the coelomocytes. We also learned that FMO3 is an essential modifier of the polyketide. FMO3 activity is essential for larval pigmentation, but in juveniles and adults, loss of FMO3 activity resulted in the animal becoming pastel purple. Linking the LC-MS analysis of this modified pigment to a naturally purple animal suggested a conserved echinochrome profile yielding a pastel purple. We interpret this result as FMO3 modifies the parent polyketide to contribute to the normal brown/green color of the animal, and that in its absence, other biochemical modifications are revealed, perhaps by other members of the large FMO family in this animal. The FMO modularity revealed here may be important in the evolutionary changes between species and for different immune challenges. We also learned that glial cells missing (GCM), a key transcription factor of the endomesoderm gene regulatory network of embryos in the sea urchin, is required for pigmentation throughout the life stages of this sea urchin, but surprisingly, is not essential for larval development, metamorphosis, or maintenance of adulthood. Mosaic knockout of either PKS or GCM revealed spatial lineage commitment in the transition from bilaterality of the larva to a pentaradial body plan of the adult. The cellular lineages identified by pigment presence or absence (wild-type or knock-out lineages, respectively) followed a strict oral/aboral profile. No circumferential segments were seen and instead we observed 10-fold symmetry in the segments of pigment expression. This suggests that the adult lineage commitments in the five outgrowths of the hydropore in the larva are early, complete, fixed, and each bilaterally symmetric. Overall, these results suggest that pigmentation of this animal is genetically determined and dependent on a population of pigment stem cells that are set-aside in a sub-region of each outgrowth of the pentaradial adult rudiment prior to metamorphosis. This study reveals the complex chemistry of pigment applicable to many organisms, and further, provides an insight into the key transitions from bilateral to pentaradial body plans unique to echinoderms.

A tumor suppressor Retinoblastoma1 is essential for embryonic development in the sea urchin.

BACKGROUND: Embryonic cells and cancer cells share various cellular characteristics important for their functions. It has been thus proposed that similar mechanisms of regulation may be present in these otherwise disparate cell types. RESULTS: To explore how regulative embryonic cells are fundamentally different from cancerous cells, we report here that a fine balance of a tumor suppressor protein Retinoblastoma1 (Rb1) and a germline factor Vasa are important for proper cell proliferation and differentiation of the somatic cells during embryogenesis of the sea urchin. Rb1 knockdown blocked embryonic development and induced Vasa accumulation in the entire embryo, while its overexpression resulted in a smaller-sized embryo with differentiated body structures. These results suggest that a titrated level of Rb1 protein may be essential for a proper balance of cell proliferation and differentiation during development. Vasa knockdown or overexpression, on the other hand, reduced or increased Rb1 protein expression, respectively. CONCLUSIONS: Taken together, it appears that Vasa protein positively regulates Rb1 protein while Rb1 protein negatively regulates Vasa protein, balancing the act of these two antagonistic molecules in somatic cells. This mechanism may provide a fine control of cell proliferation and differentiation, which is essential for regulative embryonic development.

Evolutionary modification of AGS protein contributes to formation of micromeres in sea urchins.

Evolution is proposed to result, in part, from acquisition of new developmental programs. One such example is the appearance of the micromeres in a sea urchin that form by an asymmetric cell division at the 4th embryonic cleavage and function as a major signaling center in the embryo. Micromeres are not present in other echinoderms and thus are  considered as a derived feature, yet its acquisition mechanism is unknown. Here, we report that the polarity factor AGS and its associated proteins are responsible for micromere formation. Evolutionary modifications of AGS protein seem to have provided the cortical recruitment and binding of AGS to the vegetal cortex, contributing to formation of micromeres in the sea urchins. Indeed, introduction of sea urchin AGS into the sea star embryo induces asymmetric cell divisions, suggesting that the molecular evolution of AGS protein is key in the transition of echinoderms to micromere formation and the current developmental style of sea urchins not seen in other echinoderms.

Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer.

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)-mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. SIGNIFICANCE: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype.See related commentary by Corte and Byers, p. 16.This article is highlighted in the In This Issue feature, p. 1.

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.