Title: Cellular Protrusion-Mediated Intercellular Communication during Epidermal Remodeling in Zebrafish Problem or question being addressed: Multicellular organisms rely on intercellular communication to regulate cellular behaviors during development and to maintain homeostasis. Therefore, any disruptions in the communication process can lead to developmental defects and various disorders. Previous studies have shown several major intercellular signaling mechanisms1, furthermore, local signaling is described to have a diffusion-based mechanism. In which signaling molecules from signal-sending cells are released into nearby tissues to generate a concentration gradient in order to reach their target cells. However, it is not well described how accurate and efficient, freely diffuse signaling molecules can be, when it comes to finding their targets. In addition to the diffusion-based model, several research groups, including us, have shown that cells can communicate via long cellular protrusions such as cytonemes2, tunneling nanotubes2, and airinemes2. These cellular structures make sure that morphogens from signal-sending cells are delivered precisely and efficiently to their target cells by direct contact across long distances. Moreover, our recent preliminary results showed similar cellular processes extended from fully differentiated keratinocyte cells (Fig.1), targeting undifferentiated keratinocytes in zebrafish. In this study, we will first try to find out the functional role of these keratinocyte protrusions and then look for molecular bases of signaling pathways mediated by these protrusions, and lastly, we will test if malfunctions of these protrusions (i.e., disruptions in cell-cell communication) are linked to skin diseases such as psoriasis. The rationale for your approach: The skin is the largest organ of the body, which serves as a protective barrier to protect the body from the environment, and the outermost layer of the skin, the epidermis, is mostly made up of keratinocyte cells, which play an important role in repair and homeostasis of the skin. Previous studies have shown that the differentiation and proliferation of keratinocytes post skin damage can be triggered by multiple microenvironmental factors3. However, it is not well understood how cellular protrusion-mediated local signaling controls skin barrier functions. A study done by the Carney group showed that embryonic keratinocytes are replaced by post-embryonic keratinocytes differentiated from the basal stem cell population during metamorphosis in zebrafish4, which matches time point that we see the highest frequency of the keratinocyte protrusions. Additionally, Delta-Notch signaling has been well studied for its involvement in keratinocyte differentiation5-6. Thus, we hypothesized that these keratinocyte protrusions are responsible for activating Notch signaling pathway at the keratinocyte during metamorphosis for proper keratinocyte differentiation. To test this hypothesis, we will use zebrafish, which has similar basic epidermal structures (Fig. 2) to that of mammals and provides optical transparency that allows us to visualize cell-cell interactions in real-time. Details of the suggested approach: 1) Our preliminary data suggested that periderm cells at the keratinocyte are the cell population that constantly produce protrusions. To test the functional role of these protrusions, we will block the production of these keratinocyte protrusions with a transgenic fish line that expresses the dominant negative form of Cdc42N17 (dnCdc42) in the presence of appropriate drug treatment. Cdc42 has been described in several studies for its role in cytoskeletal organization and filopodial extensions in other systems are blocked upon its inhibition7. If our prediction is correct about the involvement of these protrusions in the differentiation of the periderm keratinocytes, then we expect to see a disrupted periderm after inhibition of the protrusions. 2) To test if keratinocyte protrusions are responsible for activating Delta-Notch signaling at the keratinocyte, we will inhibit these protrusions as described in the previous step but will be comparing the expression level of notch positive cells using our Notch reporter line (TP1:eGFP) before and after the inhibition. This will provide clues for us of the involvement of these protrusions in activating the notch signaling pathway at the keratinocyte, which is likely related to keratinocyte differentiation. 3) To test if malfunctions of keratinocyte protrusions are linked with psoriasis, we will use periderm-targeted green fluorophore to label periderm cells in our psoriatic mutant fish, atp1b1a-/- and clint1a-/-, to perform overnight Timelapse imaging at metamorphic stage to monitor the behavior of keratinocyte protrusions (e.g., frequency, morphology, etc.) and compare that with our WT (wildtype) control fish. If keratinocyte protrusions are involved in the differentiation process of keratinocytes, we expect to see an increase in frequency of these protrusions. How it will affect the broader field: With this study, we will have a better understanding of how keratinocyte cells use these cellular protrusions to communicate with each other in maintaining skin barrier function, since miscommunication between skin cells can lead to skin disorders such as psoriasis. Psoriasis is a chronic inflammatory skin disease, currently with no cures, that affects approximately 125 million people worldwide and more than 8 million people in the U.S.8.Therefore, there is a need for finding cures for the disease. Psoriasis patients have built up skin cells at the epidermis due to over proliferation of keratinocyte cells9, and the cause of this hyper-proliferation is not well understood. If our hypothesis is correct, we will find more cellular protrusions extended from keratinocytes of psoriatic patients, and thus, a potential treatment for psoriasis will be to develop a drug to block the production of excess protrusions to prevent the hyper-proliferation of keratinocyte cells. Overall, our study will provide new insights into cell-cell communication that can be shared with the scientific community. References [1]Alberts B, Johnson A, Lewis J, et al. 2002 General Principles of Cell Communication. Molecular Biology of the Cell. 4th Edition. New York: Garland Science.[2]Daly CA, Hall ET, Ogden SK. 2022 Regulatory mechanisms of cytoneme-based morphogen transport. Cell. Mol. Life Sci. 79, 119.(doi:10.1007/s00018-022-04148-x)[3]Pastar I, Stojadinovic O, Yin NC, Ramirez H, Nusbaum AG, Sawaya A, Patel SB, Khalid L, Isseroff RR, Tomic-Canic M. 2014 Epithelialization in Wound Healing: A Comprehensive Review.Adv. Wound Care.3:445–464. (doi: 10.1089/wound.2013.0473)[4]Lee RT, Asharani PV, Carney TJ. 2014. Basal keratinocytes contribute to all strata of the adult zebrafish epidermis.PloS one, 9(1), e84858. (doi:10.1371/journal.pone.0084858)[5]Rangarajan A, Talora C, Okuyama R, Nicolas M, Mammucari C, Oh H, Aster JC, Krishna S, Metzger D, Chambon P, Miele L, Aguet M, Radtke F, Dotto GP. 2001 Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.The EMBO journal, 20(13), 3427–3436.(doi:10.1093/emboj/20.13.3427 [6]Moriyama M, Durham AD, Moriyama H, Hasegawa K, Nishikawa S, Radtke F, Osawa M. 2008 Multiple roles of Notch signaling in the regulation of epidermal development.Developmental cell, 14(4), 594–604.(doi:10.1016/j.devcel.2008.01.017)[7]Etienne-Manneville S. 2004 Cdc42 - the centre of polarity. Journal of Cell Science. 117:1291-1300.(doi:10.1242/jcs.01115).[8]Armstrong AW, Mehta MD, Schupp CW, Gondo GC, Bell SJ, Griffiths CEM. 2021 Psoriasis Prevalence in Adults in the United States. JAMA Dermatol.(doi:10.1001/jamadermatol.2021.2007)[9]“Psoriasis: Causes.” 25 September 2022 The National Psoriasis Foundation, https://www.psoriasis.org/causes/.