Uncommon / Non-model Organism Questionaire

Blog series organized by Eric Peterman, Michael Onken, Kristen Verhey, and Daniel M. Suter

Yelena Bernadskaya, Ph.D., Senior Research Scientist, New York University. With contribution from my summer undergraduate student, Nadia Guevara

Briefly describe the model you use.
Our lab uses the tunicate ascidian Ciona robusta to answer questions about numerous topics ranging from induction of cell fate during development, mechanics of morphogenetic cell movement, to heart regeneration. The lifecycle of Ciona includes a free-swimming larval stage, before attaching to a hard surface and undergoing metamorphosis to transition to a sessile adult (Figure 1A). Ascidians have a long history as model organisms and their chordate characteristics were identified as early as the mid 19^th century by Alexander Kowalevsky (1866) and they were instrumental in Edwin Conklin’s description of mosaic embryonic development (Conklin, 1905). Tunicates are classified as the sister group to the vertebrates, with their chordate features most apparent during their embryonic and larval stages (Figure 2). Ciona adults are hermaphrodites, producing both eggs and sperm, but are only weakly self-fertile. The embryos have a rapidly dividing invariant early cell lineage, which allows studies of
regional fate induction and cell-cell interaction, all within ~18 hours post fertilization of the eggs. The sequencing of the Ciona genome in 2002 as well as the development of electroporation-based transgenics in the 90’s, adaption of CRISPR technologies 1 and single-cell sequencing technologies^{2, 3}, has allowed Ciona to blossom into an exceptional model for developmental, systems, and cell biology. It is estimated that there are ~70 labs working on ascidians around the world⁴ .

Figure 1. Ciona robusta adult and embryonic development. A. Adult Ciona individual highlighting the sperm duct (white arrowhead) egg duct (black arrowhead) and siphons. B-D. Ciona robusta embryos shown at 6, 8, and 9 hours post fertilization (hpf) reared at 22 O C. Highlighted are membranes of epidermal cells using an epidermal specific enhancer EphB1 driving the cell membrane protein hCD4 tagged with mCherry. The nuclei and membranes of the cardiogenic B7.5 cell lineage, which includes the anterior tail muscle (ATMs) and Trunk Ventral Cells (TVCs), is highlighted with a B7.5-lineage specific enhancer Mesp driving histone H2B tagged with mCherry (red) and membrane specific hCD4::GFP. Note the mosaic inheritance of the epidermal marker in B.

Can you give a quick overview of your work and why your model organism is best suited for this work?
My work addresses how cells coordinate behavior at the individual level to produce emergent complex behaviors that contribute to morphogenesis. The Ciona robusta model is particularly useful to study collective cell behavior because of its highly simplified cardiogenic lineage which consists of just two migratory bilateral cell pairs on either side of the developing embryo, termed the Trunk Ventral Cells, (TVCs, Figure 1, 2). This has provided me with the simplest possible model of collective cell migration. Using this simplified framework, we have identified the migration of this cardiac linage as a supracellular event where the two migrating cells team up to achieve better directionality and migration speed in their tissue-dense embryonic environment 5 . This appears to be regulated at the molecular level by the ability of the cells to sense extracellular collagen through the Discoidin domain receptor (Ddr), the only receptor tyrosine kinase that binds a non-soluble ligand 6 . Thus, we are developing a biophysical pipeline through which cells sense their immediate environment and convert that information into physical collective properties that facilitate their movement through the embryo.

Figure 2. Ciona embryos reveal their chordate origins. A. Diagram of Ciona embryonic tissues at the late tailbud (9 hours post fertilization at 22O C). B. Phalloidin and DAPI staining to visualize embryonic tissues and nuclei. Images and figure generated by Nadia Guevara, research student mentored in the lab under the NYU Summer Undergraduate Research Program (SURP) in the NYU Biology Department.

Have you worked in other model systems before, and how does your current system compare to previous systems?
Throughout my career I’ve worked with two classic systems in developmental biology, Drosophila melanogaster and C. elegans. Both were instrumental in solidifying my interest in early embryonic development and genetics. The main difference between these organisms and my current Ciona model is that I no longer have to flip flies or pick worms! Our lab does not maintain Ciona strains in the lab (although transgenic strains are available for purchase from CITRES, a Japanese repository of Ciona germline transgenics). Instead, we receive a monthly shipment of wild-caught Ciona robusta from the west coast, which lasts up to two weeks in our simple aquarium set up. Another large advantage is the electroporation method of transgenesis that many ascidian labs employ. No more building double mutant strains balanced over an inversion and marked with roller (C. elegans people understand!), I select my cocktail of markers and perturbations and co-electroporate them in any combination I need. All this
adds up to a very versatile and low maintenance model that allows me to answer questions within a few weeks rather than a few months.

What are the best and most challenging parts of using your model?

When I started my work in Ciona the biggest challenge was the lack of forward genetics and lack of isogenic strains. The former is no longer an issue due to the development of efficient CRISPR techniques. The latter is offset by the large number of embryos
produced by a single electroporation protocol. The greatest advantage of the Ciona system lies in the ability to target even rare cell lineages for perturbation or expression of markers due to extensive profiling of lineage-specific cis-regulatory elements (Figure
1 B-D). Due to the small size of the Ciona genome (150-170 Mb) if we don’t already have an enhancer subcloned we can usually find one just by looking a few kb upstream of a given gene’s start site.

An interesting feature of the system is mosaic inheritance of plasmids electroporated into the one cell zygote (Figure 1B, Figure 3). While it creates an inherent variability in expression of markers or perturbations it can also be leveraged as an internal control for
clonal analysis. For example, I have used the mosaic inheritance of a marker plasmid along with perturbation of adhesion to ask how the leader/trailer states are established in the migratory cell pairs of the Ciona cardiogenic lineage. This allowed me to focus on embryos where only one cell of the pair is affected and ask how this changes the overall behavior of the migrating cell pair. I found that leader/trailer choice is made based on the relative levels of cell adhesion to the extracellular matrix⁵.

Figure 3. Mosaic inheritance of the Foxf enhancer driving mCherry in the B7.5 cardiogenic cell lineage. In 40% of electroporated embryos the marker is inherited by either the leader or the trailer, giving us the ability to perturb one cell in the
migratory cell pair. B7.5-lineage nuclei are marked using the tissue-specific Mesp>H2B::GFP transgene, which drives expression earlier and is inherited by both cells. Figure adapted from Bernadskaya et al., 2021

In your own words, can you describe the importance of using uncommon / non-model organisms in research?
I believe that our current conception of non-model organisms is the result of a recent myopia. Founders of the fields of developmental biology and genetics did not limit themselves to the study of a single organism. Thomas Hunt Morgan and Edwin Conklin all worked on a diverse array of organisms, ranging from ascidians to fiddler crabs to articulate questions about development that we are still addressing today. The Nobel laureate Eric Kandel used the giant snail Aplysia to study neuronal signal transduction and memory storage, a model that at the time was only used in two labs in France. The development of genomic tools has allowed Elaine Ostrander’s lab to use dog breeds to be used as preexisting isolated populations to study genetics of chondrodysplasia and cancer^{7, 8}. The common thread in these is the utility of the organism in answering a given question. Fiddler crabs have one large claw so they can be useful models for left/right asymmetry. Eric Kandel wanted to study neurons, so he chose a model that had large, easily accessible neurons that were invariantly positioned.

Elain Ostrander utilized pure-bred dogs for the wealth of their breeding information and bread associated characteristics to identify genes linked to developmental regulation and adult phenotypes. It behooves us to remember that the variation inherent in organisms currently bypassed for study can be leveraged to answer questions that even the best-defined model systems are cannot. With the breadth of modern techniques like whole genome sequencing and multiomics we are entering a phase where uncommon model organisms will not stay uncommon for very long.

1. Gandhi, S., Haeussler, M., Razy-Krajka, F., Christiaen, L. & Stolfi, A. Evaluation and
   rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in
   Ciona. Dev Biol 425, 8-20 (2017).

2. Cao, C. et al. Comprehensive single-cell transcriptome lineages of a proto-vertebrate. Nature 571, 349-354 (2019).
3. Wang, W. et al. A single-cell transcriptional roadmap for cardiopharyngeal fate diversification. Nat Cell Biol 21, 674-686 (2019).
4. Stolfi, A. & Brown, F.D. Tunicata, in Evolutionary Developmental Biology of Invertebrates 6: Deuterostomia. (ed. A. Wanninger) 135-204 (Springer Vienna, Vienna; 2015).
5. Bernadskaya, Y.Y., Yue, H., Copos, C., Christiaen, L. & Mogilner, A. Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells. Elife 10 (2021).
6. Bernadskaya, Y.Y., Brahmbhatt, S., Gline, S.E., Wang, W. & Christiaen, L. Discoidin-domain receptor coordinates cell-matrix adhesion and collective polarity in migratory cardiopharyngeal progenitors. Nat Commun 10, 57 (2019).
7. Evans, J.M. et al. Multi-omics approach identifies germline regulatory variants associated with hematopoietic malignancies in retriever dog breeds. PLoS Genet 17, e1009543 (2021).
8. Parker, H.G. et al. An expressed fgf4 retrogene is associated with breed-defining chondrodysplasia in domestic dogs. Science 325, 995-998 (2009).