PRIMO lab interests

Fertilization 

Species specific sperm-egg interactions are essential for sexual reproduction. Broadcast spawning of marine organisms are under particularly stringent conditions since eggs released into the water column can be exposed to multiple different sperm. Bindin isolated from the sperm acrosome resulted in insoluble particles that caused homospecific eggs to aggregate, whereas no aggregation occurred with heterospecific eggs. Therefore, Bindin was concluded to play a critical role in fertilization yet its function has never been tested. Here we report that Bindin is required for fertilization. Cas9 mediated gene inactivation in a sea urchin resulted 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.  [doi: 10.1073/pnas.2109636118]

 

Multipotent cells

As embryogenesis proceeds from a totipotent zygote into a multicellular organism, diverse cell types are specified by a progressive loss of potency. By contrast, germ cells retain the potential for totipotency and give rise to the next generation.

The juvenile sea urchin is patterned from cells that are set-aside during embryogenesis and shuttled into the larval coelomic pouches, the site of adult rudiment formation. Cells that contribute to the coelomic pouches come from various early embryonic lineages, including the small micromeres lineage. We are testing the role of Vasa, Nanos, and Piwi, three conserved germ line markers, in sea urchin development. We find that Vasa, Nanos, and Piwi are selectively expressed in the small micromere lineage during embryogenesis. As larval development proceeds, the domain of Vasa expression expands to the entire developing adult rudiment. Thus, Vasa is not a strict germ line marker in the sea urchin, but may instead indicate multipotency. Functional analysis revealed that Nanos is required to maintain the identity of small micromeres as “set-aside” cells. Nanos-depleted small micromeres ectopically divide and are not incorporated into the larval coelomic pouches. Thus, the small micromeres are likely multipotent cells that give rise to many tissues, which probably includes, but is not limited to the germ line. We hypothesize that the germline is segregated late in larval development, rather than during embryogenesis. Furthermore, we propose that the sea urchin uses a 2-step germline determination mechanism: first a long-term multipotent precursor is specified from which later the primordial germ cells (PGCs) are segregated. Our work suggests a broader role for the traditional germline genes Vasa, Nanos, and Piwi in establishing and maintaining the fate of multipotent cells in 2-step germline determination.

 

 

Vasa has multiple roles

Vasa, an ATP-dependent RNA helicase, is broadly conserved among various organisms from cnidarians to mammals. It has a rich history of utility as a germline marker, and is believed to function as a positive translational regulator in the determination and maintenance of germline cells. Studies in non-model organisms including our favorite animal sea urchins, however, revealed that Vasa is also present in somatic cells of many tissues. In many cases these cells are multipotent, are non-germline associated, and give rise to a variety of different tissue types. Our recent work demonstrated that Vasa functions in the regulation of the cell cycle during early embryogenesis of the sea urchin. Additional evidence from our ongoing research suggests that sea urchins utilize this molecule Vasa in multiple cell lineages (embryonic cell, germ cell, and adult stem cell) for multiple functions (cell cycle, germ cell specification and potentially regeneration).

 

 

Post-transcriptional regulation in small micromeres

Vasa, Nanos, and Piwi are proteins that accumulate in the sea urchin small micromeres. We have begun to test if post-transcriptional mechanisms are involved in this specific localization. These 3 proteins were also shown to be involved in translational control in different organisms: Nanos was shown to be a translational repressor, Vasa and Piwi were shown to be translational activators. We want to understand the translational functions of these 3 proteins in sea urchin small micromeres. We are now using a luciferase assay in sea urchin cell-free cap-dependent translation system to understand these mechanisms.

 

 

 

 

Translational repression by Nanos in echinoderms

Nanos is a RNA-binding protein which was first described as a translational repressor in Drosophila. The translational repression is mediated through interaction between nanos and pumilio, which binds RNAs such as hunchback and cyclin B. These target RNAs contain a conserved motif, the Nanos Response Element, in their 3’UTR. Nanos is required for the survival and maintenance of primordial germ cells during embryogenesis. Its expression is highly regulated at the RNA and protein level, any misexpression induces cell cycle and developmental defects. Nanos has been found in all animals tested. In the sea urchin, three nanos homologs are present in the genome of Strongylocentrotus purpuratus (Sp), and all of them are expressed, with differential timing, in the small micromeres (SMM). Morpholino injection against Sp nanos1 and Sp nanos2 indicates that in the sea urchin, these homologs are required for adult rudiment formation. Now, using two sea urchins, Sp and Hemicentrotus pulcherrimus (Hp), our results show that a combination of selective RNA retention, translational control, and protein stability mechanisms instills selective nanos expression in the SMM lineage.

 

 

Genetic regulation of pigmentation