The reproductive potential of an adult depends on the embryo forming specialized stem cells, called primordial germ cells. They are the only cells of the body that are able to develop into the eggs or sperm of the adult. If this mechanism fails, or if those cells are lost, the adult will not be able to make eggs or sperm, and will be reproductively sterile. The process of developing these important primordial germ cells is not understood, but they are a type of stem cells with the ultimate in developmental capability. These investigators are using a simple animal as a model to explore how the process of primordial germ cell formation works. They are using echinoderms, i.e. sea urchins and sea stars, as a model to explore this mechanism with an understanding that they will reveal general and fundamental principles of this process. Embryos from these animals are easily manipulated, they develop outside of the adult, and advanced genomic resources are available. Echinoderms are also closely related to vertebrates, so these results will have broad ramifications for animals in which this process is difficult to study.The research support will also have a significant impact beyond the bench as our lab has an aggressive outreach program. We are regular hosts to URM faculty and students, Professor Wessel serves as a scientific advisor to several preK-12 schools, and has involved a variety of students in the lab in observation, hands-on research, authorship, and creative learning. Overall, our outreach activities over the past 3 years have involved directly over 300 citizens, and benefit both the members of the lab and society, hopefully changing us all for a healthy science outlook in the future.

Project Report

Multipotent cells are present in all animals and are capable of developing into many different types of tissues. The prototypic multipotent cell includes the human hematopoietic stem cells and the I-cells of Hydra. In many animals, multipotent cells are segregated early in development and evolutionary analysis suggests that this developmental strategy is common. These early segregating cells are often the source of primordial germ cells – the cells that eventually give rise to eggs and sperm. Recent functional analysis shows that essential regulatory factors for many multipotent cells include the DEAD-box helicase vasa, the argonaute member piwi, and the zinc-finger protein nanos. Although originally recognized as specific germ cell determinants, these genes also function in stem cell development and recent results suggests that their ancestral function may have been in formation of multipotent cells (Juliano and Wessel, 2010). Through molecular, biochemical, and in vivo analyses, we seek to identify the mechanisms essential for the segregation of multipotent cells early in development, for their maintenance through early development, and for their potential to differentiate into diverse tissue types necessary to produce a reproductive adult. We use the small micromeres (SMics) of the sea urchin as a model for this process. Aim 1: Programming a multipotent cell. In the original application we proposed to identify elements enriched in the germ line, for which we found many. Surprisingly, most of these sMic enriched mRNAs are actually maternally loaded, and then selectively become enriched in the PGCs by degradation in all somatic cells of the embryo. This property appears to be the result of an element involved in mRNA turnover missing in the multipotent cells. Cnot6 is a part of the deadenylatase complex involved in mRNA turnover. It is everywhere except in the multipotent cells. This could help explain the observation made in Gustafson and Wesel, 2011, in which exogenous mRNA selectively accumulates in the sMics, but is degraded everywhere else. We have learned that CNOT6 is lost in the sMics by a nanos-dependant mechanism. The 3’UTR of the CNOT6 mRNA has two NREs and is therefore degraded only in this cell following nanos expression. Blocking nanos expression, mutating the NREs, or blocking the NREs each allows CNOT 6 mRNA accumulation and aberrant development. This work has recently been published (Swartz et al., 2014). Aim 2: Mechanism of vasa and piwi accumulation specifically in the multipotent cells by post-translational regulation. We have made substantial progress in this area by testing a variety of ways in which the E3-ligases selectively result in protein turnover. We have identified several other E3 ligases that may be involved in this regulation and are in the process of testing their role. We found 21 SOCs box containing mRNAs in the embryo and have tested accumulation profiles to help in this process. These results were recently published (Zazueta and Wessel, 2014). Like with the CNOT6 mRNA regulation, we hypothesize that Gus and perhaps other E3 ligases are regulated by nanos – mediated mRNA degradation selectively in the sMics. In the future, we will test this hypothesis by nanos knock-downs as we have for CNOT6. Aim 3: Inductive re-programming of a multipotent cell. We have been fortunate on this part of the project as well. In the screen for proteins enriched in the sMics, Z426 came out with an interesting pattern of accumulation. This is a large multi-zinc finger protein that when knocked down, leads to upregulation of Vasa in non-multipotent cells. This response is very similar to what happens when the Mics are removed, but in this case they remain intact. It is possible that Z426 is part of the signaling pathway that Mics keep active in other cells, and without it Vasa is able to accumulate to high levels and reconfigure the germ line lineage. We have emphasized progress in Aims 1 and 2 mostly and have not made as much progress in this aim. It will be a focus of future work. The intellectual merit of the proposed activity: The research integrates the disciplines of 1) developmental potency, 2) stem cells, 3) the function of classic germ-line determinants, 4) cellular signaling mechanisms, 5) post-translational gene regulation, and 6) evolutionary transitions in development. We anticipate that the results will significantly impact research in diverse areas of high priority for the NSF, and with the varied training and aggressive outreach activities of the investigator, the project will effectively impact society and its future scientists. My teaching activities have reinforced the importance of this work. Bio 1330, Biology of Reproduction enables students to explore mechanisms of germ line determination – among other topics – and this broad educational opportunity reinforced the goal of the project even further. It is fortunate to have the background from this research to help guide the educational opportunities also in the classroom.

Agency
National Science Foundation (NSF)
Institute
Division of Integrative Organismal Systems (IOS)
Application #
1120972
Program Officer
Steven L. Klein
Project Start
Project End
Budget Start
2011-08-15
Budget End
2014-07-31
Support Year
Fiscal Year
2011
Total Cost
$480,000
Indirect Cost
Name
Brown University
Department
Type
DUNS #
City
Providence
State
RI
Country
United States
Zip Code
02912