The Scf/Kit pathway implements self-organized epithelial patterning

•Xenopus epidermal multiciliated cells evenly space before radial intercalation•Pattern formation involves mutual repulsion and affinity for outer-layer cell junctions•Arp2/3-dependent actin remodeling is required patterning•The Scf/Kit pathway promotes both How global patterns emerge from individual behaviors poorly understood. In the Xenopus embryonic epidermis, (MCCs) are born in a random pattern within an inner mesenchymal layer subsequently intercalate at regular intervals into outer epithelial layer. Using video microscopy mathematical modeling, we found that emergence among motile immature MCCs toward intercellular junctions. Consistently, Arp2/3-mediated MCC patterning. Mechanistically, show Kit tyrosine kinase receptor, expressed MCCs, its ligand Scf, cells, distribution. Membrane-associated Scf behaves as potent adhesive cue while soluble form their repulsion. Finally, expression sufficient to confer order disordered heterologous population. This work reveals how single signaling system can implement self-organized large-scale Understanding molecular mechanisms forces underlying establishment of orderly cellular one long-standing aims developmental biology. Classical genetic approaches have led identification robust gene regulatory networks (GRNs) generate by concomitantly specifying identity position (Briscoe Small, 2015Briscoe J. Small S. Morphogen rules: design principles gradient-mediated embryo patterning.Development. 2015; 142: 3996-4009Crossref PubMed Scopus (234) Google Scholar; Davidson, 2010Davidson E.H. Emerging properties animal networks.Nature. 2010; 468: 911-920Crossref (377) Scholar). Regular also when stochastic movements populations partially restricted (e.g., repulsion), rat retina (Galli-Resta et al., 2002Galli-Resta L. Novelli E. Viegi A. Dynamic microtubule-dependent interactions homotypic neurones monolayered arrays during retinal development.Development. 2002; 129: 3803-3814PubMed Scholar), mouse cerebral cortex (Villar-Cerviño 2013Villar-Cerviño V. Molano-Mazón M. Catchpole T. Valdeolmillos Henkemeyer Martínez L.M. Borrell Marín O. Contact controls dispersion final distribution Cajal-Retzius cells.Neuron. 2013; 77: 457-471Abstract Full Text PDF (70) zebrafish epidermal-pigmented (Walderich 2016Walderich B. Singh A.P. Mahalwar P. Nüsslein-Volhard C. Homotypic competition regulates proliferation tiling pigment colour formation.Nat. Commun. 2016; 7: 11462Crossref (28) Drosophila hemocytes (Davis 2012Davis J.R. Huang C.Y. Zanet Harrison Rosten Cox Soong D.Y. Dunn G.A. Stramer B.M. Emergence through contact inhibition locomotion.Development. 2012; 139: 4555-4560Crossref (47) but underpinnings these processes only starting be unraveled. One example ordered epidermis amphibian Xenopus, where hundreds distributed strict spacing mucus-producing goblet (Deblandre 1999Deblandre Wettstein D.A. Koyano-Nakagawa N. Kintner A two-step mechanism generates ciliated skin embryos.Development. 1999; 126: 4715-4728Crossref The ontogeny this mucociliary epithelium multistep process (Cibois 2014Cibois Scerbo Thomé Pasini Kodjabachian Induction differentiation epidermis.in: Kloc Kubiak J.Z. Development. John Wiley & Sons, 2014Crossref (3) At cleavage stages, asymmetric divisions partition non-neural ectoderm gastrula gives rise sealed containing layer, together with osmoregulatory ionocytes, serotonin-secreting small secretory (SSCs), basal cells. Notch, Bmp, Wnt pathways control number SSCs Dubaissi 2014Dubaissi Rousseau K. Lea R. Soto X. Nardeosingh Schweickert Amaya Thornton D.J. Papalopulu type develops alongside ionocytes provides protective, anti-infective function frog epidermis.Development. 2014; 141: 1514-1525Crossref (51) Walentek 2014Walentek Bogusch Thumberger Vick Beyer Blum novel ciliary motility tadpoles.Development. 1526-1533Crossref (38) Stubbs 2006Stubbs J.L. Davidson Keller Radial intercalation 2006; 133: 2507-2515Crossref (80) Hayes 2007Hayes J.M. Kim S.K. Abitua P.B. Park T.J. Herrington E.R. Kitayama Grow M.W. Ueno Wallingford J.B. Identification ciliogenesis factors using new vivo model development.Dev. Biol. 2007; 312: 115-130Crossref (90) Quigley 2011Quigley I.K. Specification ion transport larval skin.Development. 2011; 138: 705-714Crossref (76) Cibois 2015Cibois Luxardi G. Chevalier Mercey Zaragosi L.E. Barbry Marcet BMP signalling construction vertebrate epithelia.Development. 2352-2363Crossref (32) Haas 2019Haas Gómez Vázquez Sun D.I. Tran H.T. Brislinger Tasca Shomroni Vleminckx ΔN-Tp63 mediates Wnt/beta-catenin-induced stem epithelia.Cell Rep. 2019; 28: 3338-3352.e6Abstract (16) Niehrs, 2014Huang Y.L. Niehrs Polarized ectodermal fate Xenopus.Dev. Cell. 29: 250-257Abstract (29) Starting neurula migrate apically radially they resume Strikingly, regularly mature epithelium, possibly optimize fluid flow powered beating cilia. always insert individually correspondence junctions least three (vertices) (Chung 2014Chung M.I. Kwon Tu F. Brooks Gupta Meyer Baker J.C. Marcotte E.M. Coordinated genomic movement RFX2.eLife. 3: e01439Crossref Deblandre first phase which emit polarized protrusions probe vertices, followed apical aspect emerges expands surface Sedzinski 2016Sedzinski Hannezo Biro vivo.Dev. 36: 24-35Abstract (43) Multiple studies explored 2012Kim Lake B.B. Haremaki Weinstein D.C. Sokol S.Y. Rab11 planar polarity migratory behavior epidermis.Dev. 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Fluorescent situ hybridization (FISH) embryos marker α-tubulin (α-tub) revealed stage 14 (early neural plate stage), were irregularly (Figure 1A) very often direct 1B). Co-staining antibody against tight junction ZO-1, mark showed α-tub-positive not positioned (Figures 1A 1C). course development, however, increasingly isolated 1B) localized each vertex 1C) finally inserted 1A). To quantify progression randomly distribution, developed index Delaunay triangulation centroids MCCs. inversely proportional variation distances progressively increased over 12 h elapsed 25, intercalated 1D). increase non-exclusively selective death, cell-fate reversal, or misplaced detection apoptotic executioner caspase-3/7 activity proxy apoptosis failed reveal dying embryos, opposed cycloheximide-treated (Trindade 2003Trindade Messenger Papin Grimmer D. Fairclough Tada Smith Regulation Bix3.Development. 2003; 4611-4622Crossref (20) Scholar) S1A). evaluate contribution performed α-tubFISH transgenic line expressing RFP under MCC-specific α-tub promoter (Stubbs Due long half-life (26 h), any switching different should retain fluorescence losing endogenous transcripts. was never observed, ruling out reversal normal development S1B S1C). We thus favored hypothesis plays major role pattern. motile, injected constructs carrying fluorescent markers GFP LifeActGFP (Belin 2014Belin Goins Mullins R.D. Comparative analysis tools imaging architecture.BioArchitecture. 4: 189-202Crossref (74) labeled living embryos. Spinning disk confocal GFP-expressing moved actively until eventually stopped began 2A; Video As F-actin-binding reporter allowed much better visualization shape protrusions, exploited most our subsequent experiments. could record mean instant speed 55 LifeActGFP-expressing maximum 8 h. about 0.5 μm/min 2.5 then slowed down successive 6 2B). extended retracted actin-rich lamellipodia changed flat more compact pyramidal 2C; S1B). These observations indeed important factor https://www.cell.com/cms/asset/5d6d2f4f-f71e-4fbb-9bc2-b51b7b3bcec3/mmc2.mp4Loading ... Download .mp4 (22.14 MB) Help files S1. Time-lapse motility, related Figure 2(A) move plane 2.(B) cytoskeleton 2. next built energy-based identify minimal ingredients necessary recapitulate simplified model, bi-dimensional representing Villar-Cerviño introduced term repulsion, such proximity energy. addition, reasoned homing must imply marked Thus, second affinity, overlap decreased Since two-dimensional represent regions highest possible no additional vertex-specific information needed. included steric constraint hard-core potential, prevent complete density comparable typical situations observed 14, standard Monte-Carlo scheme used allow evolution. step, given assigned random, diffusion-like prospective displacement, systematically accepted if lowered total If, contrary, energy, acceptance probability decayed exponentially energy increase. equilibrium configurations. affinity-dominated regime, relocated nearby did disperse 2D; S2A). repulsion-dominated 2E; S2B). intermediate regime terms commensurate, located scattered, although perfectly 2F; S2C), resembling normally resumed simple https://www.cell.com/cms/asset/8d2c0d90-5f52-43cd-9d12-947d5c25fc8b/mmc3.mp4Loading (11.83 S2. establishment, simulation dominated home do properly (related 2).(B) properly, 2).(C) balanced, correctly detectable vivo. First, that, whenever LifeActGFP-positive came contact, withdrew other 3A 3A′ ; S3A). Measurement pairs colliding independently collision took place, quickly decrease 3B). Colliding halt, remained 12.11 ± 18.96 min 3G), regained opposite directions 80% cases Second, preferentially along here membrane-localized (92.55% 94 analyzed closely >2 h) 3C 3H; S3B). confirmed likely https://www.cell.com/cms/asset/a46b29b6-28f9-47ad-9549-288cfa5cb168/mmc4.mp4Loading (17.6 S3. Mutual underlie 3(A) When come via lamellipodia-like structures, withdraw other, 3.α-tub::GFP tracked relative mRFP-injected Red dark blue dots lines migration path n°1 n°2, respectively (see 3A). Arrows point n°2 (white) n°3 (red), changes direction migration.(B) 3. appeared established transient contacts LifeActGFP-enriched 3A'), LifeActGFP-rich crawled dissect patterning, advantage pharmacological inhibitors main polymerization: CK-666, inhibitor actin-branching nucleator Arp2/3 (Hetrick 2013Hetrick Han M.S. Helgeson L.A. Nolen molecules CK-666 CK-869 inhibit actin-related 2/3 blocking activating conformational change.Chem. 20: 701-712Abstract (155) SMIFH2, actin-bundling Formin (Rizvi 2009Rizvi S.A. Neidt Cui Feiger Z. Skau C.T. Gardel M.L. Kozmin Kovar D.R. characterization molecule formin-mediated assembly.Chem. 2009; 16: 1158-1168Abstract (222) CK-666-treated emerged S2B) 3E, 3I, 3J). Consistent observation, S2C) 4-fold time (48.54 49.10 min) thus, forming clusters S2D; S4A). Furthermore, correlation lost S2E). However, (92.31% 26 MCCs) 3H). speed, distance covered displacement reduced compared S2F S2G). An apparent occurrence filopodia S2H S2H′; S4B), similar described ArpC3−/− murine fibroblast (Suraneni 2015Suraneni Fogelson Rubinstein Noguera Volkmann Hanein Mogilner Li leading-edge protrusion absence complex.Mol. 26: 901-912Crossref (35) observed. https://www.cell.com/cms/asset/45594fee-a6e6-41d2-a128-92733887c353/mmc5.mp4Loading (14.23 S4. requires Arp2/3-dependent remodeling, strongly 3.(B) spiky filopodia-like structures broader, ones, Conversely, treated delayed 3F, 3J agreement published data (Sedzinski Our results importance branched searched sustain attention drawn ligand/receptor couple Scf/Kit, known motility. receptor (RTK) transcriptional target MCC-inducing multicilin laevis (Kim 2018Kim Ma Shokhirev M.N. I. Multicilin activated E2F4 induce primary fibroblasts.Sci. 2018; 8: 12369Crossref (18) 2014Ma Omran H. drives centriole biogenesis E2F proteins.Genes Dev. 1461-1471Crossref 2012Stubbs Vladar E.K. Axelrod J.D. assembly multiciliate differentiation.Nat. 14: 140-147Crossref (157) Whole-mount FISH transcripts kit-L kit-S homologs specifically 4A scf-L enriched S3B, S3B′, S3C) excluded 4B). many tetrapods, synthesized transmembrane molecule, alternative splicing exon remove serine-protease site. result, exon-6-containing isoform cleaved release peptide, exon-6-spliced longer cell-membrane-associated (Brannan 1991Brannan C.I. Lyman S.D. Williams D.E. Eisenman Anderson D.M. Cosman Bedell M.A. Jenkins N.A. Copeland N.G. Steel-Dickie mutation encodes c-kit lacking cytoplasmic domains.Proc. Natl. Acad. USA. 1991; 88: 4671-4674Crossref (254) Flanagan 1991Flanagan J.G. Chan Leder Transmembrane kit growth determined missing Sld mutant.Cell. 64: 1025-1035Abstract (610) Lennartsson Rönnstrand, 2012Lennartsson Rönnstrand Stem receptor/c-Kit: basic science clinical implications.Physiol. Rev. 92: 1619-1649Crossref (440) Schol