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1. Specification of distinct cell types in a sensory-adhesive organ important for metamorphosis in tunicate larvae

   https://doi.org/10.1371/journal.pbio.3002555  

   Johnson, Christopher J; Razy-Krajka, Florian; Zeng, Fan; Piekarz, Katarzyna M; Biliya, Shweta; Rothbächer, Ute; Stolfi, Alberto (March 2024, PLOS Biology)

   Fernandez-Valverde, Selene L (Ed.)

   The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least 5 distinct cell types in the papillae of the model tunicateCiona, allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult ofCiona. 

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2. Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum

   https://doi.org/10.1186/s13227-022-00198-1  

   Bump, Paul; Khariton, Margarita; Stubbert, Clover; Moyen, Nicole E.; Yan, Jia; Wang, Bo; Lowe, Christopher J. (June 2022, EvoDevo)

   Abstract BackgroundThere are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordateSchizocardium californium(Cameron and Perez in Zootaxa 3569:79–88, 2012) to answer these questions. ResultsWe identified distinct patterns of cell proliferation between larval and adult body plan formation ofS. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. ConclusionsCell proliferation during the development ofS. californicumhas distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation ofS. californicumis mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals. 

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3. Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function

   https://doi.org/10.1242/bio.060002  

   Johnson, Christopher J; Kulkarni, Akhil; Buxton, William J; Hui, Tsz Y; Kayastha, Anusha; Khoja, Alwin A; Leandre, Joviane; Mehta, Vanshika V; Ostrowski, Logan; Pareizs, Erica G; et al (September 2023, Biology Open)

   ABSTRACT Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate ‘capstone’ research course. This small screen revealed at least one gene, Vamp1/2/3, which appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available. 

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4. A bacterial membrane-disrupting protein stimulates animal metamorphosis

   https://doi.org/10.1128/mbio.03573-24  

   Malter, Kyle E; Dunbar, Tiffany L; Westin, Carl; Darin, Emily; Alfaro, Josefa Rivera; Shikuma, Nicholas J (December 2024, mBio)

   Dubilier, Nicole (Ed.)

   ABSTRACT Diverse marine animals undergo a metamorphic larval-to-juvenile transition in response to surface-bound bacteria. Although this host-microbe interaction is critical to establishing and maintaining marine animal populations, the functional activity of bacterial products and how they activate the host’s metamorphosis program has not yet been defined for any animal. The marine bacteriumPseudoalteromonas luteoviolaceastimulates the metamorphosis of a tubeworm calledHydroides elegansby producing a molecular syringe called metamorphosis-associated contractile structures (MACs). MACs stimulate metamorphosis by injecting a protein effector termed metamorphosis-inducing factor 1 (Mif1) into tubeworm larvae. Here, we show that MACs bind to tubeworm cilia and form visible pores on the cilia membrane surface, which are smaller and less numerous in the absence of Mif1.In vitro, Mif1 associates with eukaryotic lipid membranes and possesses phospholipase activity. MACs can also deliver Mif1 to human cell lines and cause parallel phenotypes, including cell surface binding, membrane disruption, calcium flux, and mitogen-activated protein kinase activation. Finally, MACs can also stimulate metamorphosis by delivering two unrelated membrane-disrupting proteins, MLKL and RegIIIɑ. Our findings demonstrate that membrane disruption by MACs and Mif1 is necessary forHydroidesmetamorphosis, connecting the activity of a bacterial protein effector to the developmental transition of a marine animal. IMPORTANCEThis research describes a mechanism wherein a bacterium prompts the metamorphic development of an animal from larva to juvenile form by injecting a protein that disrupts membranes in the larval cilia. Specifically, results show that a bacterial contractile injection system and the protein effector it injects form pores in larval cilia, influencing critical signaling pathways like mitogen-activated protein kinase and calcium flux, ultimately driving animal metamorphosis. This discovery sheds light on how a bacterial protein effector exerts its activity through membrane disruption, a phenomenon observed in various bacterial toxins affecting cellular functions, and elicits a developmental response. This work reveals a potential strategy used by marine organisms to respond to microbial cues, which could inform efforts in coral reef restoration and biofouling prevention. The study’s insights into metamorphosis-associated contractile structures’ delivery of protein effectors to specific anatomical locations highlight prospects for future biomedical and environmental applications. 

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5. Molecular mechanisms of tubulogenesis revealed in the sea star hydro-vascular organ

   https://doi.org/10.1038/s41467-023-37947-2  

   Perillo, Margherita; Swartz, S Zachary; Pieplow, Cosmo; Wessel, Gary M (December 2023, Nature Communications)

   Abstract A fundamental goal in the organogenesis field is to understand how cells organize into tubular shapes. Toward this aim, we have established the hydro-vascular organ in the sea starPatiria miniataas a model for tubulogenesis. In this animal, bilateral tubes grow out from the tip of the developing gut, and precisely extend to specific sites in the larva. This growth involves cell migration coupled with mitosis in distinct zones. Cell proliferation requires FGF signaling, whereas the three-dimensional orientation of the organ depends on Wnt signaling. Specification and maintenance of tube cell fate requires Delta/Notch signaling. Moreover, we identify target genes of the FGF pathway that contribute to tube morphology, revealing molecular mechanisms for tube outgrowth. Finally, we report that FGF activates the Six1/2 transcription factor, which serves as an evolutionarily ancient regulator of branching morphogenesis. This study uncovers distinct mechanisms of tubulogenesis in vivo and we propose that cellular dynamics in the sea star hydro-vascular organ represents a key comparison for understanding the evolution of vertebrate organs. 

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