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The ion channel Anoctamin 10/TMEM16K coordinates organ morphogenesis across scales in the urochordate notochord [1]

['Zonglai Liang', 'Michael Sars Centre', 'University Of Bergen', 'Bergen', 'Daniel Christiaan Dondorp', 'Marios Chatzigeorgiou']

Date: 2024-08

During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na + /Ca 2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca 2+ signaling molecular modules that regulate notochord morphogenesis and Ca 2+ dynamics. In addition, we find that the highly conserved Ca 2+ sensors calmodulin (CaM) and Ca 2+ /calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca 2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the “encoding” and “decoding” of developmental Ca 2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales.

Data Availability: All individual quantitative observations that underlie the data summarized in the figures and results of our paper can be found in Zenodo: 10.5281/zenodo.12506448 . Raw image of gel shown in S4A Fig is also located in 10.5281/zenodo.12506448 . Code used to process confocal data and quantify the distance to skeleton metric has been deposited in Zenodo: 10.5281/zenodo.5539439 . The Mesmerize GitHub repo with the code used to perform Ca2+ imaging analysis is deposited in Zenodo: 10.5281/zenodo.5539439 .

In addition, using time-lapse imaging and translational fusions we show that LOF of Ano10 alters the localization of RhoA and cofilin, interfering with cytoskeletal organization and affecting cell motility and shape. We further demonstrate that Ano10 acts not as a phospholipid scramblase but instead as an ion channel, where its role in establishing and/or maintaining an appropriate electrochemical balance across cell membranes is likely essential for notochord morphogenesis.

Here, we leverage the simplicity and genetic accessibility of the C. intestinalis notochord to study the role of C. intestinalis Ano10/Tmem16k (gene model: KH.C3.109/KY21.Chr3.1036 from here on referred to as Ano10) in tube morphogenesis. We employ tissue-specific CRISPR/Cas9 knockout and rescue experiments to reveal that Ano10 is required for convergent extension, lumen expansion, and lumen connection. By combining genome editing and translational fusions, we show that Ano10 is required for the subcellular localization of CaM and CaMK. Using volumetric in vivo functional imaging and the Ca 2+ integrator CAMPARI, we show that LOF of Ano10 perturbs Ca 2+ signaling during notochord morphogenesis. Exploiting genetically encoded calcium sponges, genome editing, and pharmacological perturbations, we show that Ca 2+ signaling machinery in the ER and PM orchestrated by Ano10 contributes to convergent extension and tubulogenesis.

Anoctamins generally function as Ca 2+ -activated Cl - channels (CaCCs) and phospholipid scramblases (PS); the latter capable of translocating phospholipids between the 2 monolayers of a membrane [ 40 , 41 , 47 ]. They have been reported to take part in diverse cellular functions, including for example signal transduction, cell migration, Cl - secretion, and volume regulation [ 41 , 43 , 48 , 49 ]. While some Anoctamins, such as ANO1/TMEM16A, act as a CaCC [ 40 , 50 , 51 ], and ANO6/TMEM16F as a PS [ 47 ]; for other members of the family, including ANO10/TMEM16K, their functions remain under fruitful debate [ 41 , 52 , 53 ]. In particular, the subcellular localization and function of several Anoctamins including ANO10/TMEM16K has been debated with some studies reporting, for example, an exclusively intracellular localization for ANO10/TMEM16K with no evidence of activity as a channel [ 54 – 56 ], while others have reported both detectable plasma membrane (PM) localization (albeit with higher portion of localization in intracellular compartments) and channel activity [ 57 – 59 ].

To further focus on how bioelectrical signaling shapes the developing tubular organ, we turned to transmembrane pumps, transporters, and ion channels. Of these, the role of the widely conserved Anoctamin (Ano/Tmem16) family proteins [ 38 , 39 ] is essentially unexplored in developmental tube morphogenesis even though several of the Anoctamins have previously been shown to be expressed in mammalian tubular structures including pancreatic acinar cells, proximal renal tubules, and several glands (e.g., submandibular glands, Leydig cells) [ 40 – 46 ].

Tubulogenesis has been intensively studied across a diversity of models, such as the salivary gland of Drosophila, the excretory cells of Caenorhabditis elegans, the lungs, mammary glands, and neural tube of mice and the notochord of zebrafish and C. intestinalis [ 3 , 22 – 24 ]. Transmembrane proteins have since emerged as an important regulator of signal transduction during the process [ 22 , 25 – 29 ]. More generally transmembrane proteins such as ion channels and transporters are important in signaling during development [ 29 – 37 ].

The notochord, a defining feature of all chordates, is a tubular organ located along the embryonic midline region of embryos [ 7 , 8 ]. Formation of this flexible rod, tapered at both ends [ 9 ], is fundamental to providing structural support to the developing chordate embryo. In the case of vertebrates, the notochord further serves as a signaling center that secretes factors to pattern surrounding tissues [ 10 ]. Ascidians, which belong to the sister group to vertebrates the tunicates, have a notochord composed of only 40 cells [ 11 – 13 ]. The small cell number makes it an ideally tractable model for dissecting the rudimentary cellular and molecular mechanisms underlying the multistep process of notochord formation. For example, in Ciona intestinalis (formerly known as C. intestinalis type B), notochord development begins with cell intercalation-driven convergent extension [ 14 – 16 ]. Next, the notochord elongates to form a cylindrical rod via actomyosin network dependent-cell shape changes [ 17 , 18 ]. Fundamental to tube formation is the establishment of lumens. Here, the notochord cells of Ciona undergo a mesenchymal-to-epithelial transition (MET) that leads to the emergence of apical domains at the opposite ends of each cell [ 11 , 19 ]. Extracellular lumens thereby appear and then expand between neighboring cells of the developing tube [ 17 , 19 – 21 ]. Finally, the notochord cells crawl bidirectionally, exhibiting an endothelial-like cell morphology, which leads to merging of the lumens [ 11 , 19 ].

Biological tubes play an essential role in embryogenesis, organogenesis, and postembryonic physiology [ 1 , 2 ]. Indeed, tubular structures are widespread across vertebrates and invertebrates, including salivary glands, renal tubules, vasculature, intestinal tract, and bronchial tubules. Reliable formation of these diverse tubular organs depends on the highly coordinated interplay between collective cell behaviors and complex signaling processes [ 1 , 3 – 5 ]. Errors in tube formation during development or malfunctions in adult tubular structures can result in severe pathologies [ 2 , 6 ].

Organ morphogenesis is a complex biological process that involves the coordinated behavior of cells in space and time to give rise to functional biological form. Elucidating the fundamental mechanisms that coordinate organ morphogenesis across multiple scales, from the molecular to the supracellular levels, will have profound implications for a large range of biological problems in health and disease.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002762

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