Jia L. Song

Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.

Select microRNAs are essential for early development in the sea urchin

microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and can serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. miRNAs as well as a large population of putative piRNAs (piwi-interacting RNAs) had dynamic accumulation profiles through early development. Defects in morphogenesis caused by loss of Drosha could be rescued with four miRNAs. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.

Genes involved in the RNA interference pathway are differentially expressed during sea urchin development

RNA‐mediated interference (RNAi) is a conserved gene silencing mechanism that involves double‐stranded RNA as a signal to trigger the sequence‐specific degradation of target mRNA, resulting in posttranscriptional silencing and/or translational repression. Bioinformatic searches in the sea urchin genome database identified homologs of Drosha, DGCR5, Dicer, TRBP, Exportin‐5, and Argonautes. Quantitative, real‐time polymerase chain reaction indicated that all mRNA accumulate in eggs and in variable levels throughout early development. Whole‐mount in situ RNA hybridization showed that all of the important players of the RNAi silencing pathway have abundant mRNA accumulation in oocytes and eggs, but have distinct spatial and temporal expression patterns throughout development. Sequence analysis revealed that each of the four Argonautes examined contain conserved residues important for RNAseH activity within the Piwi domain. This study elucidated that genes involved in the RNAi silencing pathway have dynamic expression and, thus, may have regulatory roles during germ cell development and embryogenesis. Developmental Dynamics 236:3180–3190, 2007.

Function and regulation of microRNA-31 in development and disease

MicroRNAs (miRNAs) are small noncoding RNAs that orchestrate numerous cellular processes both under normal physiological conditions as well as in diseases. This review summarizes the functional roles and transcriptional regulation of the highly evolutionarily conserved miRNA, microRNA‐31 (miR‐31). miR‐31 is an important regulator of embryonic implantation, development, bone and muscle homeostasis, and immune system function. Its own regulation is disrupted during the onset and progression of cancer and autoimmune disorders such as psoriasis and systemic lupus erythematosus. Limited studies suggest that miR‐31 is transcriptionally regulated by epigenetics, such as methylation and acetylation, as well as by a number of transcription factors. Overall, miR‐31 regulates diverse cellular and developmental processes by targeting genes involved in cell proliferation, apoptosis, cell differentiation, and cell motility. Mol. Reprod. Dev. 83: 654–674, 2016

Piwi regulates Vasa accumulation during embryogenesis in the sea urchin

Background: Piwi proteins are essential for germ line development, stem cell maintenance, and more recently found to function in epigenetic and somatic gene regulation. In the sea urchin Strongylocentrotus purpuratus, two Piwi proteins, Seawi and Piwi‐like1, have been identified, yet their functional contributions have not been reported. Results: Here we found that Seawi protein was localized uniformly in the early embryo and then became enriched in the primordial germ cells (PGCs) (the small micromere lineage) from blastula stage and thereafter. Morpholino knockdown of Sp‐seawi diminished PGC‐specific localization of Seawi proteins, and altered expression of other germ line markers such as Vasa and Gustavus, but had no effect on Nanos. Furthermore, Seawi knockdown transiently resulted in Vasa positive cell proliferation in the right coelomic pouch that appear to be derived from the small micromere lineage, yet they quickly disappeared with an indication of apoptosis by larval stage. Severe Seawi knockdown resulted in an increased number of apoptotic cells in the entire gut area. Conclusion: Piwi proteins appear to regulate PGC proliferation perhaps through control of Vasa accumulation. In this organism, Piwi is likely regulating mRNAs, not just transposons, and is potentially functioning both inside and outside of the germ line during embryogenesis. Developmental Dynamics 243:451–458, 2014.

The forkhead transcription factor FoxY regulates Nanos.

FoxY is a member of the forkhead transcription factor family that appeared enriched in the presumptive germ line of sea urchins (Ransick et al. Dev Biol 2002;246:132). Here, we test the hypothesis that FoxY is involved in germ line determination in this animal. We found two splice forms of FoxY that share the same DNA‐binding domain, but vary in the carboxy‐terminal trans‐activation/repression domain. Both forms of the FoxY protein are present in the egg and in the early embryo, and their mRNAs accumulate to their highest levels in the small micromeres and adjacent non‐skeletogenic mesoderm. Knockdown of FoxY resulted in a dramatic decrease in Nanos mRNA and protein levels as well as a loss of coelomic pouches in 2‐week‐old larvae. Our results indicate that FoxY positively regulates Nanos at the transcriptional level and is essential for reproductive potential in this organism. Mol. Reprod. Dev. 79: 680–688, 2012.

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

microRNA-31 modulates skeletal patterning in the sea urchin embryo.

MicroRNAs (miRNAs) are small non-coding RNAs that repress the translation and reduce the stability of target mRNAs in animal cells. microRNA-31 (miR-31) is known to play a role in cancer, bone formation and lymphatic development. However, studies to understand the function of miR-31 in embryogenesis have been limited. We examined the regulatory role of miR-31 in early development using the sea urchin as a model. miR-31 is expressed at all stages of development and its knockdown (KD) disrupts the patterning and function of primary mesenchyme cells (PMCs), which form the embryonic skeleton spicules. We identified that miR-31 directly represses Pmar1, Alx1, Snail and VegfR7 within the PMC gene regulatory network using reporter constructs. Further, blocking the miR-31-mediated repression of Alx1 and/or VegfR7 in the developing embryo resulted in defects in PMC patterning and skeletogenesis. The majority of the mislocalized PMCs in miR-31 KD embryos did not express VegfR10, indicating that miR-31 regulates VegfR gene expression within PMCs. In addition, miR-31 indirectly suppresses Vegf3 expression in the ectoderm. These results indicate that miR-31 coordinately suppresses genes within the PMCs and in the ectoderm to impact PMC patterning and skeletogenesis. This study identifies the novel function and molecular mechanism of miR-31-mediated regulation in the developing embryo. Summary: In sea urchin, miRNA-31 acts in primary mesenchyme cells to inhibit components of the gene regulatory network that promotes skeletogenesis.

The small GTPase Arf6 regulates sea urchin morphogenesis

The small GTPase Arf6 is a conserved protein that is expressed in all metazoans. Arf6 remodels cytoskeletal actin and mediates membrane protein trafficking between the plasma membrane in its active form and endosomal compartments in its inactive form. While a rich knowledge exists for the cellular functions of Arf6, relatively little is known about its physiological role in development. This study examines the function of Arf6 in mediating cellular morphogenesis in early development. We dissect the function of Arf6 with a loss-of-function morpholino and constitutively active Arf6-Q67L construct. We focus on the two cell types that undergo active directed migration: the primary mesenchyme cells (PMCs) that give rise to the sea urchin skeleton and endodermal cells that form the gut. Our results indicate that Arf6 plays an important role in skeleton formation and PMC migration, in part due to its ability to remodel actin. We also found that embryos injected with Arf6 morpholino have gastrulation defects and embryos injected with constitutively active Arf6 have endodermal cells detached from the gut epithelium with decreased junctional cadherin staining, indicating that Arf6 may mediate the recycling of cadherin. Thus, Arf6 impacts cells that undergo coordinated movement to form embryonic structures in the developing embryo.