Research

PRIMO lab interests

Research:

Our research focus is on the mechanisms of making eggs. Our approaches include the entire life cycle of the organism from when the primordial germ cells are first made, to how the gonad forms, and to how the eggs are made as a single cell developmental machine. We use diverse approaches: Cas9-based modifications, single cell RNA-seq analysis, accelerated animal husbandry, confocal microscopy, and many genomic resources to interrogate this process. Our favorite organisms for this work are echinoderms, yielding large numbers of eggs amenable to broad manipulations. Remarkably, echinoderms do not exhibit reproductive aging or senescence as is seen in many animals, including humans, and we use this unusual character, as well as comparative analyses, to leverage our analyses. Please join us on this research conversation!

Comparative single cell RNA-seq of echinoderm gonads 

Echinoderms keep producing functional gametes throughout their lifespan, in some cases exceeding 200 years. This suggests an active set of stem cells that are constantly cycling in an out of seasonal reproductive growth. Here, we present single cell RNA sequencing (scRNAseq) datasets of mature ovaries from two sea urchin species (Strongylocentrotus purpuratus and Lytechinus variegatus) and one sea star species (Patiria miniata). We link key genes with cell clusters with in situ hybridizations in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production. It is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum. 

tSNE plots representing the scRNA seq dataset of Strongylocentrotus purpuratus, Lytechinus variegatus and Patiria miniata ovaries.

Oulhen N, Morita S, Pieplow C, Onorato TM, Foster S, Wessel G. Conservation and contrast in cell states of echinoderm ovaries. Mol Reprod Dev. 2023 Dec 6. doi: 10.1002/mrd.23721. Epub ahead of print. PMID: 38054259.

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]

Genetic regulation of pigmentation 

Highlights:

1. Color variations of the adult sea urchin are genetically determined and independent of nutrition or environment.
2. Color variations of pigment are created by downstream modifications of a single pigment pathway and LC-MS profiles reveal a pigment palate for purple color.
3. Color patterning of the adult sea urchin is established independently within each compartment of the pentaradial adult structure along the oral-aboral axis.
4. A pigment stem cell population is set-aside in each bilateral outgrowth of the pentaraidal adult rudiment prior to metamorphosis and this positional information is strictly retained into adulthood.

Echinoderms display a vast array of pigmentation and patterning in larval and adult life stages. This coloration is thought to be important for immune defense and camouflage. However, neither the cellular nor molecular mechanism that regulates this complex coloration in the adult is known. Here we knocked out three different genes thought to be involved in the pigmentation pathway(s) of larvae and grew the embryos to adulthood. The genes tested were polyketide synthase (PKS), Flavin-dependent monooxygenase family 3 (FMO3) and glial cells missing (GCM). We found that disabling of the PKS gene at fertilization resulted in albinism throughout all life stages and throughout all cells and tissues of this animal, including the immune cells of the coelomocytes. We also learned that FMO3 is an essential modifier of the polyketide. FMO3 activity is essential for larval pigmentation, but in juveniles and adults, loss of FMO3 activity resulted in the animal becoming pastel purple. Linking the LC-MS analysis of this modified pigment to a naturally purple animal suggested a conserved echinochrome profile yielding a pastel purple. We interpret this result as FMO3 modifies the parent polyketide to contribute to the normal brown/green color of the animal, and that in its absence, other biochemical modifications are revealed, perhaps by other members of the large FMO family in this animal. The FMO modularity revealed here may be important in the evolutionary changes between species and for different immune challenges. We also learned that glial cells missing (GCM), a key transcription factor of the endomesoderm gene regulatory network of embryos in the sea urchin, is required for pigmentation throughout the life stages of this sea urchin, but surprisingly, is not essential for larval development, metamorphosis, or maintenance of adulthood. Mosaic knockout of either PKS or GCM revealed spatial lineage commitment in the transition from bilaterality of the larva to a pentaradial body plan of the adult. The cellular lineages identified by pigment presence or absence (wild-type or knock-out lineages, respectively) followed a strict oral/aboral profile. No circumferential segments were seen and instead we observed 10-fold symmetry in the segments of pigment expression. This suggests that the adult lineage commitments in the five outgrowths of the hydropore in the larva are early, complete, fixed, and each bilaterally symmetric. Overall, these results suggest that pigmentation of this animal is genetically determined and dependent on a population of pigment stem cells that are set-aside in a sub-region of each outgrowth of the pentaradial adult rudiment prior to metamorphosis. This study reveals the complex chemistry of pigment applicable to many organisms, and further, provides an insight into the key transitions from bilateral to pentaradial body plans unique to echinoderms.  [doi: 10.1038/s41598-020-58584-5]

Measuring an oocyte transcriptome by its polar body

Much of early animal development is driven by mRNAs, proteins, mitochondria and other maternally deposited materials in the oocyte. This maternal endowment is essential in most cases for success of an embryo in early development. We are interested in what mRNAs are present in oocytes, and the variation found between individual oocytes because those differences may be able to predict the range of possible developmental outcomes even when considering the oocytes from the same female. We measure the transcriptomes of single cells (oocytes or polar bodies biopsied from the oocyte), within the same genotype (in the case of mice) or between genotypes (different mouse backgrounds or human IVF/ART patients). The biological variability of transcriptomes can be quantified between single cells within a genotype and the comparison between genotypes can reveal genes that are differentially expressed in a robust manner. We also demonstrated that detection and quantification of mRNA in human polar bodies is possible and reflects the transcript profile of the MII oocyte. The quantification of mRNAs is of particular importance since some transcripts have highly variable expression between oocytes, this variance is reflected in the polar body, and these variations may explain differences in developmental outcome.

All four oocyte samples show a high degree of overlap with each other. The total number of genes for each sample is shown in parentheses, and the total number of all genes in all four samples is equal to 12,708 genes. 66.7% of all genes were detected in at least three of the four oocyte samples, and 50.0% of all genes were detected in all four oocyte samples. b, the larger circle represents the total number of genes in each of the four oocyte samples, and the smaller circle shows the overlap of the four sibling polar bodies. The percentage was calculated by taking the total number of genes shared between the sibling oocyte and polar body and dividing by the total number of genes in the polar body. c, the overlaps of genes transcribed in sibling oocyte and PB samples among the four independent comparisons are represented as described in a. In total, there were 4,973 genes found between all the overlap data sets and 279 genes that were sampled in all eight samples. Of the 4,973 overlap gene set, ∼46% were detected in at least two of the overlaps.

Reich A, Klatsky P, Carson S, Wessel G. The transcriptome of a human polar body accurately reflects its sibling oocyte. J Biol Chem. 2011 Nov 25;286(47):40743-9. doi: 10.1074/jbc.M111.289868. Epub 2011 Sep 27. PMID: 21953461; PMCID: PMC3220517. 

How to grow a biological tube.

Many biological organs arise from nascent tubular structures. The vertebrate heart, and its many chambers, is a great example of the power of the tube! Organisms make tubular structures for an enormous variety of developmental fates. We use the water vascular system in the sea star larva to examine mechanisms used to make such important tubular structures. What we have learned impacts our general understanding of such structures, and their embellishments, in many other organisms. Key steps in this process include physical constraints on the replicating cells by the basal lamina, cell signaling molecules for directional growth, specialized cellular motility within an epithelium, and expression of transcription factors in select aspects of the tubes. The clarity in this larva, and the many manipulations feasible, drive widespread progress. 

a Phylogenetic relationships of the main bilaterian groups. b Summary of sea star larval development with a focus on the hydro-vascular organ (in magenta). The hydro-vascular organ comes from mesodermal precursors located at the tip of the growing gut in gastrula (G). Tubulogenesis starts in the late gastrula (LG), continues in the early larva (EL) and larval stages (L1 to L3). In larval stages, the initial tubes that form the organ elongate posteriorly. The left tube forms the hydropore canal, an opening toward the outside environment. The organ is fully grown in late larva (LL), when the left and right tubes merge to form the closed system of the hydro-vascular organ. c Summary of the main larval tissues and organs. d–d” Transversal view of the tube trough the hydropore canal showing that the epithelium is polarized with actin on the apical side of the cells (facing the lumen) and laminin on the cell basal side. Dotted lines indicate the polarity of a single tube cell. Scale bars 10 μm. 

Perillo, M., Swartz, S.Z., Pieplow, C. et al. Molecular mechanisms of tubulogenesis revealed in the sea star hydro-vascular organ. Nat Commun 14, 2402 (2023). https://doi.org/10.1038/s41467-023-37947-2

Germline determination 

We rely on evolutionary changes to teach us about mechanisms of cell fate decisions in the germline. Germ cells and reproduction are often widely diverse, and here we used to sea urchins to explore this diversity. Using single cell RNA-seq analysis of early embryos we find that the germ cell factor Nanos is expressed more distinctly than other genes in comparing the two species. Its regulation in expression is also distinct between the species. More over, post-transcriptional and post-translational processes, dominate Nanos expression.   

Morita S, Oulhen N, Foster S, Wessel GM. Elements of divergence in germline determination in closely related species. iScience. 2023 Mar 15;26(4):106402. doi: 10.1016/j.isci.2023.106402. PMID: 37020963; PMCID: PMC10068562.

Optimization of Cas9 utilization in echinoderms

We test a variety of approaches, reagents, and protocols to enhance use of Cas9 in echinoderms. This animal is wonderfully tractable for such manipulations and with many visual phenotypes possible, analysis can be accelerated. 

Oulhen N, Pieplow C, Perillo M, Gregory P, Wessel GM. Optimizing CRISPR/Cas9-based gene manipulation in echinoderms. Dev Biol. 2022 Oct;490:117-124. doi: 10.1016/j.ydbio.2022.07.008. Epub 2022 Jul 30. PMID: 35917936.