Eric A. Gustafson

Vasa genes: emerging roles in the germ line and in multipotent cells.

Sexually reproducing metazoans establish a cell lineage during development that is ultimately dedicated to gamete production. Work in a variety of animals suggests that a group of conserved molecular determinants act in this germ line maintenance and function. The most universal of these genes are Vasa and Vasa‐like DEAD‐box RNA helicase genes. However, recent evidence indicates that Vasa genes also function in other cell types, distinct from the germ line. Here we evaluate our current understanding of Vasa function and its regulation during development, addressing Vasas emerging role in multipotent cells. We also explore the evolutionary diversification of the N‐terminal domain of this gene and how this impacts the association of Vasa with nuage‐like perinuclear structures.

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.

Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis

Vasa is a broadly conserved DEAD-box RNA helicase associated with germ line development and is expressed in multipotent cells in many animals. During embryonic development of the sea urchin Strongylocentrotus purpuratus, Vasa protein is enriched in the small micromeres despite a uniform distribution of vasa transcript. Here we show that the Vasa coding region is sufficient for its selective enrichment and find that gustavus, the B30.2/SPRY and SOCS box domain gene, contributes to this phenomenon. In vitro binding analyses show that Gustavus binds the N-terminal and DEAD-box portions of Vasa protein independently. A knockdown of Gustavus protein reduces both Vasa protein abundance and its propensity for accumulation in the small micromeres, whereas overexpression of the Vasa-interacting domain of Gustavus (GusΔSOCS) results in Vasa protein accumulation throughout the embryo. We propose that Gustavus has a conserved, positive regulatory role in Vasa protein accumulation during embryonic development.

DEAD-box helicases: posttranslational regulation and function.

Helicases are enzymes that can separate duplex oligonucleotides in a NTP-dependent fashion and are essential in all aspects of DNA and RNA metabolism. Amino acid sequence analysis identified several conserved sequence motifs in DNA and RNA helicases allowing their classification into 5 major groups (Super families SF1–SF5) [1]. DExD/H helicases share eight conserved sequence motifs, whereas the DEAD box helicase subgroup has an additional ninth conserved sequence motif [2]. These sequence motifs encompass an approximately 300–400 amino acid core region involved in ATP-binding/hydrolysis and RNA binding (Part 2: Figure 1A). Structural analyses of several DEAD-box proteins show this core region forms two RecA-like globular domains [2]. Open in a separate window Figure 1 Human DEAD-box protein (DDX) posttranslational modifications and protein-protein interactions A schematic representation of the DDX protein domain architecture features the conserved central DEAD-box core region along with the unique flanking N-terminal and C-terminal regions. Above shows the number of posttranslational documented in Table 1 with respect to their location within the DDX protein. Below lists known DDX-binding proteins, as well as where the interaction occurs on their respective DDX open reading frame.

The biology of the germ line in echinoderms.

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. Mol. Reprod. Dev. 81: 679–711, 2014.

TAF4b is Required for Mouse Spermatogonial Stem Cell Development

Long‐term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal‐enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b‐deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b‐deficient mouse testis. Here, we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age‐matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild‐type testes, TAF4b‐deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b‐deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self‐renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development. Stem Cells 2015;33:1267–1276

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.

Exogenous RNA is selectively retained in the small micromeres during sea urchin embryogenesis

The small micromeres are multipotent cells of the sea urchin embryo that populate the coelomic pouches and contribute to various tissues of the adult (Juliano and Wessel 2010). This lineage forms by two sequential unequal cleavages, divides only once prior to gastrulation, and accumulates several mRNAs and proteins selectively, including vasa, nanos, and piwi. In manipulating this embryo for experimentation, investigators often inject mRNAs to over-express proteins, thought to occur uniformly throughout the embryo and to thereby influence cells equally. We show here a new character of the small micromeres, which is a selective retention of injected synthetic mRNA. Vasa mRNA is present uniformly throughout the early embryo, but the Vasa protein accumulates selectively in the small micromeres (Voronina et al. 2008) by differential protein turnover (Gustafson et al., submitted). Here we test the ability of protein derived from exogenous mRNA to phenocopy endogenous Vasa protein by microinjecting mRNA from a vasa-GFP construct containing Xenopus β-globin UTRs. These reporter constructs were generated from the transcriptional vector pSP6T4, commonly used in sea urchin overexpression analyses (Lepage and Gache 2004). The Vasa 1F construct contains the sequence encoding the full length vasa open reading frame, and indeed, the protein from this mRNA injection accumulates selectively in the small micromeres, and eventually more in the small micromeres in the left coelomic pouch, just as the endogenous protein (Voronina et al. 2008). Co-injecting mRNA (using the same UTRs and transcriptional vector) that encodes mCherry shows fluorescence uniformly throughout the early embryo (data not shown) but later also selectively in the small micromeres. To test if either of these selective protein accumulations results from differential retention of the mRNA in small micromeres, we performed in situ hybridization on the injected embryos using sequences specific to the mRNA encoding the fluorescent proteins. Indeed, by gastrulation both the Vasa 1F-GFP and the mCherry mRNAs were retained selectively by the small micromeres. Vasa is an RNA helicase that is thought to be involved in translational regulation. In testing whether it is involved in the mRNA retention phenomenon, we manipulated the vasa sequence such that it lacks its N-terminus or its catalytic domains; both regions are required for Vasa function. Following microinjection of these mRNAs and culture of the embryos, each of the vasa mRNAs and the mCherry mRNA showed persistent and specific retention in the small micromeres. The mechanism of this selective mRNA retention is unknown, but is unlikely due to a lack of mRNA turnover mechanisms in this relatively quiescent cell since several endogenous mRNAs are turned over during development in this same cell. Since the small micromeres appear to be a multipotent cell similar to those found in other organisms, especially in the lophotrochozoans (Juliano and Wessel, 2010), this phenomenon of selective mRNA retention may reflect a shared character of multipotency.

TAF4b Regulates Oocyte-Specific Genes Essential for Meiosis.

TAF4b is a gonadal-enriched subunit of the general transcription factor TFIID that is implicated in promoting healthy ovarian aging and female fertility in mice and humans. To further explore the potential mechanism of TAF4b in promoting ovarian follicle development, we analyzed global gene expression at multiple time points in the human fetal ovary. This computational analysis revealed coordinate expression of human TAF4B and critical regulators and effectors of meiosis I including SYCP3, YBX2, STAG3, and DAZL. To address the functional relevance of this analysis, we turned to the embryonic Taf4b-deficient mouse ovary where, for the first time, we demonstrate, severe deficits in prophase I progression as well as asynapsis in Taf4b-deficient oocytes. Accordingly, TAF4b occupies the proximal promoters of many essential meiosis and oogenesis regulators, including Stra8, Dazl, Figla, and Nobox, and is required for their proper expression. These data reveal a novel TAF4b function in regulating a meiotic gene expression program in early mouse oogenesis, and support the existence of a highly conserved TAF4b-dependent gene regulatory network promoting early oocyte development in both mice and women.

Meiotic Gene Expression Initiates During Larval Development in the Sea Urchin

Background: Meiosis is a unique mechanism in gamete production and a fundamental process shared by all sexually reproducing eukaryotes. Meiosis requires several specialized and highly conserved genes whose expression can also identify the germ cells undergoing gametogenic differentiation. Sea urchins are echinoderms, which form a phylogenetic sister group of chordates. Sea urchin embryos undergo a feeding, planktonic larval phase in which they construct an adult rudiment prior to metamorphosis. Although a series of conserved meiosis genes (e.g., dmc1, msh5, rad21, rad51, and sycp1) is expressed in sea urchin oocytes, we sought to determine when in development meiosis would first be initiated. Results: We surveyed the expression of several meiotic genes and their corresponding proteins in the sea urchin Strongylocentrotus purpuratus. Surprisingly, meiotic genes are highly expressed not only in ovaries but beginning in larvae. Both RNA and protein localizations strongly suggest that meiotic gene expression initiates in tissues that will eventually give rise to the adult rudiment of the late larva. Conclusions: These results demonstrate that broad expression of the molecules associated with meiotic differentiation initiates prior to metamorphosis and may have additional functions in these cells, or mechanisms repressing their function, until later in development when gametogenesis begins. Developmental Dynamics, 2012.