ABSTRACT
Delivering molecular tools into oocytes is essential for developmental and reproductive biology. Microinjection, the conventional method, is equipment-intensive, often technically challenging, and low-yield, and is impractical in species with delicate oocytes or restricted spawning seasons. To overcome these limitations, we developed VitelloTag, a cost-effective, high-throughput system using vitellogenin-derived fusion proteins to enable efficient cargo delivery via receptor-mediated endocytosis. We demonstrate its utility by delivering Cas9/sgRNA complexes in two distantly related species for gene knockout.
ABSTRACT
Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.
Subject(s)
DEAD-box RNA Helicases/metabolism , Germ Cells/cytology , Sea Urchins/embryology , Starfish/embryology , Ubiquitin-Protein Ligases/metabolism , Animals , CRISPR-Cas Systems/genetics , Embryo Culture Techniques , Embryo, Nonmammalian/embryology , Protein Processing, Post-TranslationalABSTRACT
Specification of the primordial germ cells (PGCs) is essential for sexually reproducing animals. Although the mechanisms of PGC specification are diverse between organisms, the RNA binding protein Nanos is consistently required in the germ line in all species tested. How Nanos is selectively expressed in the germ line, however, remains largely elusive. We report that in sea urchin embryos, the early expression of Nanos2 in the PGCs requires the maternal Wnt pathway. During gastrulation, however, Nanos2 expression expands into adjacent somatic mesodermal cells and this secondary Nanos expression instead requires Delta/Notch signaling through the forkhead family member FoxY. Each of these transcriptional regulators were tested by chromatin immunoprecipitation analysis and found to directly interact with a DNA locus upstream of Nanos2. Given the conserved importance of Nanos in germ line specification, and the derived character of the micromeres and small micromeres in the sea urchin, we propose that the ancestral mechanism of Nanos2 expression in echinoderms was by induction in mesodermal cells during gastrulation.
Subject(s)
Gastrulation/physiology , Gene Expression Regulation, Developmental/physiology , RNA-Binding Proteins/metabolism , Strongylocentrotus purpuratus/embryology , Transcription, Genetic/physiology , Wnt Signaling Pathway/physiology , Animals , Germ Cells/cytology , Germ Cells/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Mesoderm/cytology , Mesoderm/embryology , Receptors, Notch/metabolism , Strongylocentrotus purpuratus/cytologyABSTRACT
Stem cells in animals often exhibit a slow cell cycle and/or low transcriptional activity referred to as quiescence. Here, we report that the translational activity in the primordial germ cells (PGCs) of the sea urchin embryo (Strongylocentrotus purpuratus) is quiescent. We measured new protein synthesis with O-propargyl-puromycin and L-homopropargylglycine Click-iT technologies, and determined that these cells synthesize protein at only 6% the level of their adjacent somatic cells. Knockdown of translation of the RNA-binding protein Nanos2 by morpholino antisense oligonucleotides, or knockout of the Nanos2 gene by CRISPR/Cas9 resulted in a significant, but partial, increase (47%) in general translation specifically in the PGCs. We found that the mRNA of the translation factor eEF1A is excluded from the PGCs in a Nanos2-dependent manner, a consequence of a Nanos/Pumilio response element (PRE) in its 3'UTR. In addition to eEF1A, the cytoplasmic pH of the PGCs appears to repress translation and simply increasing the pH also significantly restores translation selectively in the PGCs. We conclude that the PGCs of this sea urchin institute parallel pathways to quiesce translation thoroughly but transiently.
Subject(s)
Cell Cycle , Germ Cells/cytology , Protein Biosynthesis , Strongylocentrotus purpuratus/cytology , Strongylocentrotus purpuratus/metabolism , Animals , Base Sequence , Blastula/cytology , Blastula/metabolism , CRISPR-Cas Systems/genetics , Cell Cycle/genetics , Gene Expression Regulation, Developmental , Germ Cells/metabolism , Hydrogen-Ion Concentration , Mitochondria/metabolism , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/metabolism , Protein Biosynthesis/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Strongylocentrotus purpuratus/geneticsABSTRACT
A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.
Subject(s)
Exoribonucleases/metabolism , Gene Expression Regulation, Developmental , Germ Cells/cytology , Strongylocentrotus purpuratus/embryology , Animals , Base Sequence , Cell Differentiation , Cell Separation , Flow Cytometry , Gene Expression Profiling , Molecular Sequence Data , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Strongylocentrotus purpuratus/enzymology , Time Factors , TranscriptomeABSTRACT
Indirect development, in which embryogenesis gives rise to a larval form, requires that some cells retain developmental potency until they contribute to the different tissues in the adult, including the germ line, in a later, post-embryonic phase. In sea urchins, the coelomic pouches are the major contributor to the adult, but how coelomic pouch cells (CPCs) are specified during embryogenesis is unknown. Here we identify the key signaling inputs into the CPC specification network and show that the forkhead factor foxY is the first transcription factor specifically expressed in CPC progenitors. Through dissection of its cis-regulatory apparatus we determine that the foxY expression pattern is the result of two signaling inputs: first, Delta/Notch signaling activates foxY in CPC progenitors; second, Nodal signaling restricts its expression to the left side, where the adult rudiment will form, through direct repression by the Nodal target pitx2. A third signal, Hedgehog, is required for coelomic pouch morphogenesis and institution of laterality, but does not directly affect foxY transcription. Knockdown of foxY results in a failure to form coelomic pouches and disrupts the expression of virtually all transcription factors known to be expressed in this cell type. Our experiments place foxY at the top of the regulatory hierarchy underlying the specification of a cell type that maintains developmental potency.
Subject(s)
Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental/physiology , Morphogenesis/physiology , Multipotent Stem Cells/cytology , Nodal Protein/metabolism , Receptors, Notch/metabolism , Strongylocentrotus purpuratus/embryology , Animals , Fluorescent Antibody Technique , Gene Expression Regulation, Developmental/genetics , Hedgehog Proteins/metabolism , In Situ Hybridization , Morpholinos/genetics , Multipotent Stem Cells/metabolism , PhalloidineABSTRACT
The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ-line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed.
Subject(s)
Echinodermata/embryology , Embryonic Induction/physiology , Gametogenesis/physiology , Gene Expression Regulation, Developmental/physiology , Germ Cells/physiology , Models, Biological , Signal Transduction/physiology , Animals , Asymmetric Cell Division/physiology , Calcium/metabolism , Calcium Channels, L-Type/metabolism , DEAD-box RNA Helicases/metabolism , Endoplasmic Reticulum/metabolism , Species SpecificityABSTRACT
The germline of multicellular animals is segregated from somatic tissues, which is an essential developmental process for the next generation. Although certain ecdysozoans and chordates segregate their germline during embryogenesis, animals from other taxa segregate their germline after embryogenesis from multipotent progenitor cells. An overlapping set of genes, including vasa, nanos and piwi, operate in both multipotent precursors and in the germline. As we propose here, this conservation implies the existence of an underlying germline multipotency program in these cell types that has a previously underappreciated and conserved function in maintaining multipotency.
Subject(s)
Germ Cells/cytology , Animals , Embryonic Development/genetics , Embryonic Development/physiology , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Germ Cells/metabolism , Guanosine Monophosphate/metabolism , Phylogeny , Sea Urchins/embryologyABSTRACT
A fundamental goal in the organogenesis field is to understand how cells organize into tubular shapes. Toward this aim, we have established the hydro-vascular organ in the sea star Patiria miniata as a model for tubulogenesis. In this animal, bilateral tubes grow out from the tip of the developing gut, and precisely extend to specific sites in the larva. This growth involves cell migration coupled with mitosis in distinct zones. Cell proliferation requires FGF signaling, whereas the three-dimensional orientation of the organ depends on Wnt signaling. Specification and maintenance of tube cell fate requires Delta/Notch signaling. Moreover, we identify target genes of the FGF pathway that contribute to tube morphology, revealing molecular mechanisms for tube outgrowth. Finally, we report that FGF activates the Six1/2 transcription factor, which serves as an evolutionarily ancient regulator of branching morphogenesis. This study uncovers distinct mechanisms of tubulogenesis in vivo and we propose that cellular dynamics in the sea star hydro-vascular organ represents a key comparison for understanding the evolution of vertebrate organs.
Subject(s)
Cell Nucleus Division , Starfish , Animals , Cell Differentiation , Cell Movement , Starfish/genetics , Wnt Signaling PathwayABSTRACT
The early embryos of sea urchins and other echinoderms have served as experimental models for the study of cell division since the nineteenth century. Their rapid development, optical clarity, and ease of manipulation continue to offer advantages for studying spindle assembly and cytokinesis. In the absence of transgenic lines, alternative strategies must be employed to visualize microtubules and actin. Here, we describe methods to visualize actin and microtubule using either purified, recombinant proteins, or probes in in vitro-transcribed mRNAs.
Subject(s)
Microtubules , Mitosis , Animals , Germ Cells , Meiosis , Microtubules/metabolism , Sea Urchins , Spindle Apparatus/metabolismABSTRACT
Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) transition, resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI/MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after MI. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.
Subject(s)
Meiosis , Oocytes/physiology , Phosphoric Monoester Hydrolases/metabolism , Starfish/genetics , Animals , Phosphorylation , Starfish/enzymologyABSTRACT
The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes.1 However, the mechanisms that generate cellular asymmetries within the oocyte and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled (Dvl) is a cytoplasmic Wnt pathway effector required for axis development in diverse species,2-4 but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dvl localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially regulated Dvl dynamics.
Subject(s)
Body Patterning , Starfish , Animals , Embryonic Development , Oocytes/metabolism , SolubilityABSTRACT
Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.
Subject(s)
Centromere Protein A/metabolism , Centromere/metabolism , Muscle, Skeletal/metabolism , Nucleosomes/metabolism , Animals , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Division , Cell Line , Cell Proliferation , Centromere/ultrastructure , Epigenesis, Genetic , Female , Green Fluorescent Proteins/metabolism , Humans , Male , Meiosis , Mice , Mice, Inbred C57BL , Oocytes/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/metabolism , Starfish/metabolism , Testis/metabolism , Polo-Like Kinase 1ABSTRACT
With few exceptions, all animals acquire the ability to produce eggs or sperm at some point in their life cycle. Despite this near-universal requirement for sexual reproduction, there exists an incredible diversity in germ line development. For example, animals exhibit a vast range of differences in the timing at which the germ line, which retains reproductive potential, separates from the soma, or terminally differentiated, nonreproductive cells. This separation may occur during embryonic development, after gastrulation, or even in adults, depending on the organism. The molecular mechanisms of germ line segregation are also highly diverse, and intimately intertwined with the overall transition from a fertilized egg to an embryo. The earliest embryonic stages of many species are largely controlled by maternally supplied factors. Later in development, patterning control shifts to the embryonic genome and, concomitantly with this transition, the maternally supplied factors are broadly degraded. This chapter attempts to integrate these processes--germ line segregation, and how the divergence of germ line and soma may utilize the egg to embryo transitions differently. In some embryos, this difference is subtle or maybe lacking altogether, whereas in other embryos, this difference in utilization may be a key step in the divergence of the two lineages. Here, we will focus our discussion on the echinoderms, and in particular the sea urchins, in which recent studies have provided mechanistic understanding in germ line determination. We propose that the germ line in sea urchins requires an acquisition of maternal factors from the egg and, when compared to other members of the taxon, this appears to be a derived mechanism. The acquisition is early--at the 32-cell stage--and involves active protection of maternal mRNAs, which are instead degraded in somatic cells with the maternal-to-embryonic transition. We collectively refer to this model as the Time Capsule method for germ line determination.
Subject(s)
Embryo, Nonmammalian/physiology , Ovum/physiology , Animals , Biological Evolution , Embryonic Development , Gene Regulatory Networks , GenomeABSTRACT
Isolating cells based on specific gene expression enables a focused biochemical and molecular analysis. While cultured cells and hematopoietic cells, for example, are routinely isolated by fluorescence activated cell sorting (FACS), early embryonic cells are a relatively untapped source for FACS applications often because the embryos of many animals are quite limiting. Furthermore, many applications require genetic model organisms in which cells can be labeled by fluorescent transgenes, or antibodies against cell surface antigens. Here we define conditions in the sea urchin embryo for isolation of embryonic cells based on expression of specific proteins. We use the sea urchin embryo for which a nearly unlimited supply of embryonic cells is available and demonstrate the conditions for separation of the embryo into single cells, fixation of the cells for antibody penetration into the cells, and conditions for FACS of a rare cell type in the embryo. This protocol may be adapted for analysis of mRNA, chromatin, protein, or carbohydrates and depends only on the probe availability for the cell of interest. We anticipate that this protocol will be broadly applicable to embryos of other species.
Subject(s)
Blastula/cytology , Sea Urchins/cytology , Animals , Cell Separation , Flow Cytometry , Tissue Culture TechniquesABSTRACT
Members of the Vasa family of helicases are specifically localized to germ line lineages in embryos of many animal groups and, in some cases, have been shown to be required for germ line formation. Despite considerable attention to the embryology of gastropod molluscs, the germ line has not been identified in the early cleavage stages of these embryos. We have cloned a Vasa ortholog in the snail Ilyanassa and examined the distribution of IoVasa mRNA during early cleavage. Initially, the transcript is present in all cells and non-specifically localized to centrosomes in a subset of cells. The IoVasa mRNA becomes progressively more enriched in the dorsal quadrant of the embryo, and then becomes restricted to particular cells in the 4d lineage. At the 64-cell stage, IoVasa mRNA is detected in 4dL11, 4dL12, 4dR11, and 4dR12. Following another round of division in the 4d lineage, the mRNA is restricted to two cells: 4dL121 and 4dR121. By the 108-cell stage, IoVasa mRNA is no longer detectable. Because the germ line is thought to arise from the 4d lineage in spiralians, these data are consistent with the hypothesis that the Ilyanassa germ line is marked by inheritance of IoVasa and derived from the cells 4dL121 and 4dR121. Alternatively, IoVasa may be required in somatic lineages where it is expressed, and the germ line may be specified later in development.