The skin, the largest organ, functions as a primary defense mechanism. Epidermal stem cells supply undifferentiated keratinocytes that differentiate as they migrate toward the outermost skin layer. Although such a replenishment process is disrupted in various human skin diseases, its underlying mechanisms remain elusive. With high-resolution live imaging and in vivo manipulations, we revealed that Notch signaling between keratinocytes is mediated by signaling filopodia called cytonemes and is essential for proper keratinocyte differentiation and proliferation. Inhibiting keratinocyte cytonemes reduced Notch expression within undifferentiated keratinocytes, leading to abnormal differentiation and hyperproliferation, resembling human skin disease phenotypes. Overproduction of Interleukin (IL)-17 signal, associated with skin diseases like psoriasis , induces psoriatic phenotypes via cytonemes in zebrafish. Our study suggests that intercellular signaling between keratinocytes through cytonemes is critical for epidermal maintenance, and its misregulation could be an origin of human skin diseases.

Specialized cellular protrusions facilitate local intercellular communication in various species, including mammals. Among these, airinemes play a crucial role in pigment pattern formation in zebrafish by mediating long-distance Notch signaling between pigment cells. Remarkably, airinemes exhibit large vesicle-like structures at their tips, which are pulled by macrophages and delivered to target cells. The interaction between macrophages and Delta-ligand-carrying airineme vesicles is essential for initiating airineme-mediated signaling, yet the molecular details of this interaction remain elusive. Through high-resolution live imaging, genetic in vivo manipulations, and in vitro adhesion assays, we found that adhesive interactions via the extracellular domain of CD44, a class I transmembrane glycoprotein, between macrophages and airineme vesicles are critical for airineme signaling. Mutants lacking the extracellular domain of CD44 lose their adhesiveness, resulting in a significant reduction in airineme extension and pigment pattern defects. Our findings provide valuable insights into the role of adhesive interactions between signal-sending cells and macrophages in long-range intercellular signaling.

Intercellular communication is indispensable across multicellular organisms, and any aberration in this process can give rise to significant anomalies in developmental and homeostatic processes. Thus, a comprehensive understanding of its mechanisms is imperative for addressing human health-related concerns. Recent advances have expanded our understanding of intercellular communication by elucidating additional signaling modalities alongside established mechanisms. Notably, cellular protrusion-mediated long-range communication, characterized by physical contact through thin and elongated cellular protrusions between cells involved in signal transmission and reception, has emerged as a significant intercellular signaling paradigm. This chapter delves into the exploration of a signaling cellular protrusion termed 'airinemes,' discovered in the zebrafish skin. It covers their identified signaling roles and the cellular and molecular mechanisms that underpin their functionality.

Dae Seok Eom spoke with Cell Reports about his scientific journey, inspirations to become a scientist, and his work on cellular protrusions called "airinemes" that are involved in long-range intercellular signal transmission in zebrafish; in particular, he discussed recent work regarding characterization of a macrophage population that plays a critical role in this process.

The skin, the largest organ, functions as a primary defense mechanism. Epidermal stem cells supply undifferentiated keratinocytes that differentiate as they migrate toward the outermost skin layer. Although such a replenishment process is disrupted in various human skin diseases, its underlying mechanisms remain elusive. With high-resolution live imaging and in vivo manipulations, we revealed that Notch signaling between keratinocytes is mediated by signaling filopodia called cytonemes and is essential for proper keratinocyte differentiation and proliferation. Inhibiting keratinocyte cytonemes reduces Notch within undifferentiated keratinocytes, leading to abnormal differentiation and hyperproliferation, resembling human skin disease phenotypes. Overproduction of Interleukin (IL)-17 signal, associated with skin diseases like psoriasis, induces psoriatic phenotypes via cytonemes in zebrafish. Clathrin interactor 1 (clint1), crucial for epidermal homeostasis in zebrafish and humans, regulates cytoneme extension. Our study suggests that intercellular signaling between keratinocytes via cytonemes is critical for epidermal maintenance, and its misregulation could be an origin of human skin diseases.

Tissue-resident macrophages are heterogeneous and perform location-dependent functions. Skin resident macrophages play intriguing roles in long-distance intercellular signaling by mediating cellular protrusions called airinemes in zebrafish. These macrophages relay signaling molecules containing airineme vesicles between pigment cells, and their absence disrupts airineme-mediated signaling and pigment pattern formation. It is unknown if the same macrophages control both these signaling and typical immune functions or if a separate subpopulation functions in intercellular communication. With high-resolution imaging and genetic ablation approaches, we identify a macrophage subpopulation responsible for airineme-mediated signaling. These seem to be distinct from conventional skin-resident macrophages by their ameboid morphology and faster or expansive migratory behaviors. They resemble ectoderm-derived macrophages termed metaphocytes. Metaphocyte ablation markedly decreases airineme extension and signaling. In addition, these ameboid/metaphocytes require matrix metalloproteinase-9 for their migration and airineme-mediated signaling. These results reveal a macrophage subpopulation with specialized functions in airineme-mediated signaling, which may play roles in other aspects of intercellular communication.

Dae Seok Eom spoke with Cell Reports about his scientific journey, inspirations to become a scientist, and his work on cellular protrusions called "airinemes" that are involved in long-range intercellular signal transmission in zebrafish; in particular, he discussed recent work regarding characterization of a macrophage population that plays a critical role in this process.

In addition to diffusive signals, cells in tissue also communicate via long, thin cellular protrusions, such as airinemes in zebrafish. Before establishing communication, cellular protrusions must find their target cell. Here, we demonstrate that the shapes of airinemes in zebrafish are consistent with a finite persistent random walk model. The probability of contacting the target cell is maximized for a balance between ballistic search (straight) and diffusive search (highly curved, random). We find that the curvature of airinemes in zebrafish, extracted from live-cell microscopy, is approximately the same value as the optimum in the simple persistent random walk model. We also explore the ability of the target cell to infer direction of the airineme’s source, finding that there is a theoretical trade-off between search optimality and directional information. This provides a framework to characterize the shape, and performance objectives, of non-canonical cellular protrusions in general.

The gut visceral musculature plays essential roles in not only moving substances through the lumen but also maintaining the function and physiology of the gut. Although the development of the visceral musculature has been studied in multiple model organisms, how it degenerates is poorly understood. Here, we employ the Drosophila midgut as a model to demonstrate that the visceral musculature is disrupted by intrinsic and extrinsic factors, such as aging, feeding, chemical-induced tissue damage, and oncogenic transformation in the epithelium. Notably, we define four prominent visceral musculature disruption phenotypes, which we refer as ‘sprout’, ‘discontinuity’, ‘furcation’, and ‘crossover’ of the longitudinal muscle. Given that the occurrence of these phenotypes is increased during aging and under various stresses, we propose that these phenotypes can be used as quantitative readouts of deterioration of the visceral musculature. Intriguingly, administration of a tissue-damaging chemical dextran sulfate sodium (DSS) induced similar visceral musculature disruption phenotypes in zebrafish larvae, indicating that ingestion of a tissue-damaging chemical can disrupt the visceral musculature in a vertebrate as well. Our study provides insights into the deterioration of the gut visceral musculature and lays a groundwork for investigating the underlying mechanisms in Drosophila as well as other animals.

In addition to diffusive signals, cells in tissue also communicate via long, thin cellular protrusions, such as airinemes in zebrafish. Before establishing communication, cellular protrusions must find their target cell. Here we demonstrate airinemes in zebrafish are mathematically consistent with a finite persistent random walk model. The probability of contacting the target cell is maximized for a balance between ballistic search (straight) and diffusive (highly curved, random) search. We find that the curvature of airinemes in zebrafish, extracted from live cell microscopy, is approximately the same value as the optimum in the simple mathematical model.

SummaryTumors often secrete wasting factors associated with atrophy and degeneration of host tissues. If tumors were affected by the wasting factors, mechanisms allowing tumors to evade the adverse effects of the wasting factors must exist and impairing such mechanisms may attenuate tumors. We usedDrosophilamidgut tumor models to show that tumors upregulate Wingless (Wg) to oppose the growth-impeding effects caused by the wasting factor, ImpL2 (Insulin-like growth factor binding protein (IGFBP)-related protein). Growth of Yorkie (Yki)-induced tumors is dependent on Wg while either elimination ofImpL2or elevation of Insulin/IGF signaling in tumors revokes this dependency. Notably, Wg augmentation could be a general mechanism for supporting the growth of tumors with elevated ImpL2 and exploited to attenuate muscle degeneration during wasting. Our study elucidates the mechanism by which tumors negate the action of ImpL2 and implies that targeting the Wnt/Wg pathway might be an efficient treatment strategy for cancers with elevated IGFBPs.

Adhesive interactions are essential for tissue patterning and morphogenesis yet difficult to study owing to functional redundancies across genes and gene families. A useful system in which to dissect roles for cell adhesion and adhesion-dependent signaling is the pattern formed by pigment cells in skin of adult zebrafish, in which stripes represent the arrangement of neural crest derived melanophores, cells homologous to melanocytes. In a forward genetic screen for adult pattern defects, we isolated the pissarro (psr) mutant, having a variegated phenotype of spots, as well as defects in adult fin and lens. We show that psr corresponds to junctional adhesion protein 3b (jam3b) encoding a zebrafish orthologue of the two immunoglobulin-like domain receptor JAM3 (JAM-C), known for roles in adhesion and signaling in other developing tissues, and for promoting metastatic behavior of human and murine melanoma cells. We found that zebrafish jam3b is expressed post-embryonically in a variety of cells including melanophores, and that jam3b mutants have defects in melanophore survival. Jam3b supported aggregation of cells in vitro and was required autonomously by melanophores for an adherent phenotype in vivo. Genetic analyses further indicated both overlapping and non-overlapping functions with the related receptor, Immunoglobulin superfamily 11 (Igsf11) and Kit receptor tyrosine kinase. These findings suggest a model for Jam3b function in zebrafish melanophores and hint at the complexity of adhesive interactions underlying pattern formation.

In addition to diffusive morphogens, cells in developing tissues also communicate via recently discovered cellular protrusions, such as airinemes, which mediate long distance signaling between pigment cells in zebrafish. Diffusive morphogens are characterized by a diffusion coefficient. In contrast, cellular protrusions are characterized by a velocity and an angular diffusion coefficient (or equivalently a directional persistence length) that determine the airinemes' curvature. What determines these parameters? Before establishing communication, an airineme must find its target cell. Here we present a simple mathematical model of airineme target cell search. We find that the probability of contacting the target cell is maximized for a balance between ballistic search (straight airinemes) and diffusive (highly curved, random) search. Further, we extract the curvature of airinemes in zebrafish from high-resolution microscopy and use maximum likelihood estimation to extract the angular diffusion coefficient. Surprisingly, we find that it is approximately the same value as the one found in the simple mathematical model for optimal search. We are extending the model to incorporate more realistic features, including obstacles and multiple target cells.

Understanding the mechanisms of cell-to-cell communication is one of the fundamental questions in biology and medicine. In particular, long-range signalling where cells communicate over several cell diameters is vital during development and homeostasis. The major morphogens, their receptors and intracellular signalling cascades have largely been identified; however, there is a gap in our knowledge of how such signalling factors are propagated over a long distance. In addition to the diffusion-based propagation model, new modalities of disseminating signalling molecules have been identified. It has been shown that cells can communicate with direct contact through long, thin cellular protrusions between signal sending and receiving cells at a distance. Recent studies have revealed a type of cellular protrusion termed ‘airinemes’ in zebrafish pigment cell types. They share similarities with previously reported cellular protrusions; however, they also exhibit distinct morphology and features. Airinemes are indispensable for pigment pattern development by mediating long-distance Delta-Notch signalling between different pigment cell types. Notably, airineme-mediated signalling is dependent on skin-resident macrophages. Key findings of airineme-mediated intercellular signalling in pattern development, their interplay with macrophages and their implications for the understanding of cellular protrusion-mediated intercellular communication will be discussed.

Macrophages have diverse functions in immunity as well as in development and homeostasis. We identified a function for these cells in long-distance communication during postembryonic tissue remodeling. Ablation of macrophages in zebrafish prevented melanophores from coalescing into adult pigment stripes. Melanophore organization depends on signals provided by cells of the yellow xanthophore lineage via airinemes, long filamentous projections with vesicles at their tips. We show that airineme extension from originating cells, as well as vesicle deposition on target cells, depend on interactions with macrophages. These findings identify a role for macrophages in relaying long-range signals between nonimmune cells. This signaling modality may function in the remodeling and homeostasis of other tissues during normal development and disease.

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Changes in gene activity are essential for evolutionary diversification. Yet, elucidating the cellular behaviors that underlie modifications to adult form remains a profound challenge. We use neural crest-derived adult pigmentation of zebrafish and pearl danio to uncover cellular bases for alternative pattern states. We show that stripes in zebrafish require a novel class of thin, fast cellular projection to promote Delta-Notch signaling over long distances from cells of the xanthophore lineage to melanophores. Projections depended on microfilaments and microtubules, exhibited meandering trajectories, and stabilized on target cells to which they delivered membraneous vesicles. By contrast, the uniformly patterned pearl danio lacked such projections, concomitant with Colony stimulating factor 1-dependent changes in xanthophore differentiation that likely curtail signaling available to melanophores. Our study reveals a novel mechanism of cellular communication, roles for differentiation state heterogeneity in pigment cell interactions, and an unanticipated morphogenetic behavior contributing to a striking difference in adult form. https://doi.org/10.7554/eLife.12401.001 eLife digest Animals have very different patterns of skin pigmentation, and these patterns can be important for survival and reproduction. Zebrafish, for example, have horizontal dark and light stripes along their bodies, while a closely related fish called the pearl danio has an almost uniform pattern. The dark stripes of the zebrafish contain cells called melanophores, while the lighter regions contain two other types of cells known as xanthophores and iridophores. These pigment cell types interact with each other to create stripes. The iridophores establish the lighter stripes and specify the position and orientation of the dark stripes. They also produce a protein called Csf1, which allows the xanthophores to mature. As the stripes form, melanophores present in lighter stripes move into nearby dark stripes. Pearl danios also contain these three types of pigment cells, but these cells remain intermingled giving the fish their uniform color. Eom et al. have now used microscopy to image pigment cells in zebrafish and pearl danio to uncover how interactions between these cells differ in species with different pigment patterns. The technique involved tagging pigment cells with fluorescent markers and using time-lapse imaging to track them during the formation of the adult pigmentation pattern. The experiments show that stripes form in zebrafish because the cells that make the xanthophores form long, thin projections that extend to neighboring melanophores. These so-called ‘airinemes’ deliver materials to melanophores and help to clear the melanophores from interstripe regions, partly by activating a cell communication pathway called Delta-Notch signaling. These cell projections are mostly absent from the cells that make xanthophores in the pearl danio due to differences in when Csf1 is produced. This alters the timing of when the xanthophores develop, leading to the loss of long-distance airineme signaling. Eom et al.’s findings identify a new way in which cells can communicate and an unanticipated cell behavior that contributes to a striking difference in the pigmentation patterns of zebrafish and pearl danio. Future studies should further our understanding of these unique projections and reveal whether they are produced by other types of cells. https://doi.org/10.7554/eLife.12401.002 Introduction Genes contributing to phenotypic diversification are beginning to be identified, yet the morphogenetic mechanisms by which changes in gene activities are translated into species differences in form remain virtually unknown. Holding great promise for identifying such mechanisms are the diverse and ecologically important pigment patterns of fishes (Endler, 1980; Houde, 1997; Wang et al., 2006; Engeszer et al., 2008; Price et al., 2008; Seehausen et al., 2008; Roberts et al., 2009; Kelley et al., 2013) because pigment cell behaviors are observable as phenotypes unfold and because pigment pattern development is amenable to both experimental manipulation and theoretical modeling (Painter et al., 1999; Kondo and Miura, 2010; Miyazawa et al., 2010; Caballero et al., 2012; Yamanaka and Kondo, 2014; Bullara and De Decker, 2015; Volkening and Sandstede, 2015). In this regard, fishes of the genus Danio should be especially useful as their adult pigment patterns differ markedly among species and mechanisms of pattern formation are starting to be understood in zebrafish, D. rerio (Parichy, 2015; Parichy and Spiewak, 2015; Singh and Nusslein-Volhard, 2015; Watanabe and Kondo, 2015). Zebrafish have dark stripes of black melanophores with light interstripes of yellow-orange xanthophores and iridescent iridophores (Figure 1A), all of which are derived from the neural crest, either directly, or through stem cell intermediates (Budi et al., 2011; Dooley et al., 2013; Mahalwar et al., 2014; McMenamin et al., 2014; Singh et al., 2014). Interactions among pigment cells are essential to pattern formation (Watanabe and Kondo, 2015). Iridophores differentiate in the prospective interstripe, and specify the positions and orientations of melanophore stripes (Frohnhofer et al., 2013; Patterson and Parichy, 2013). Iridophores also promote the differentiation of xanthophores in the interstripe by expressing Colony stimulating factor-1 (Csf1)(Patterson and Parichy, 2013). Subsequently, interactions between cells of melanophore and xanthophore lineages are required during a period of stripe consolidation, in which some melanophores initially in the interstripe join the stripes, and, simultaneously, the stripe borders become increasingly organized (Parichy et al., 2000; Maderspacher and Nusslein-Volhard, 2003; Parichy and Turner, 2003a, 2003b; Quigley et al., 2005; Takahashi and Kondo, 2008). Finally, this pattern is reiterated as additional iridophores invade the stripes, ultimately emerging on the other side, where they terminate the first stripes and initiate new interstripes and stripes dorsally and ventrally (Patterson et al., 2014; Singh et al., 2014). Figure 1 with 3 supplements see all Download asset Open asset Pigment cell projections. (A) Zebrafish and pearl danio. Right, melanophores and xanthophores (arrows) after epinephrine treatment to contract pigment granules. (B) Long projections by zebrafish aox5+ cells of xanthophore lineage (arrows) with membraneous vesicles (arrowhead, inset). (C) Zebrafish aox5+ cells were more likely to extend projections than pearl, especially during early stripe development [7–8 SSL (Parichy et al., 2009); species x stage, χ2=103.4, d.f.=4, p<0.0001; N=929, 1259 cells for zebrafish and pearl; projections per cell: χ2=45.3, d.f.=1, p<0.0001]. (D) In zebrafish, projections were often long and fast. Bars indicate median ± interquartile range (IQR). (E) Extension and retraction (arrow) and release of vesicle (arrowhead) in zebrafish but not pearl. Scale bars: 5 mm (A, left); 50 µm (A, right); 10 µm (B); 50 µm (E). https://doi.org/10.7554/eLife.12401.003 Despite an increasing appreciation for the phenomenology of interactions among zebrafish pigment cells through cell transplantation, genetic analyses, laser ablation, in vitro manipulations, and theoretical modeling (Maderspacher and Nusslein-Volhard, 2003; Nakamasu et al., 2009; Eom et al., 2012; Yamanaka and Kondo, 2014), the cellular and molecular mechanisms relevant to these interactions in vivo remain largely unknown. Moreover, the ways in which such interactions may have changed during the development of naturally occurring, alternative pattern states across species have yet to be explored. Interesting in this context is pearl danio, D. albolineatus, which has a nearly uniform pattern of fewer melanophores, more xanthophores and an intermingling of all three pigment cell classes (Figure 1A) (Quigley et al., 2005; Mills et al., 2007; Patterson et al., 2014; McCluskey and Postlethwait, 2015). Here, we show by time-lapse imaging of cells in their native tissue environment that stripe consolidation in zebrafish requires a novel class of fast, long cellular projection, extended by cells of the xanthophore lineage to melanophores. These projections, which we call 'airinemes,' contribute to transducing a Delta-Notch signal that promotes the clearance of melanophores from the developing interstripe. We further show that production and targeting of airinemes are differentiation-state dependent. Finally, using interspecific cell transplantation and transgenic manipulations we demonstrate that evolution of the very different, uniform pattern of pearl danio has entailed the loss of long-distance airineme signaling, concomitant with modifications to xanthophore differentiation. Our results provide novel insights relevant to empirical and theoretical understanding of pigment pattern formation in zebrafish, as well as mechanisms of cellular communication and how they change during the evolution of adult phenotypes. Results Fast projections are extended frequently by cells of the xanthophore lineage in zebrafish but not pearl danio To determine if species differences are associated with modifications to pigment cell interactions, we visualized cells using membrane-targeted fluorophores and time-lapse imaging during adult pigment pattern formation (Budi et al., 2011). In zebrafish, cells of the xanthophore lineage, marked by expression of aldehyde oxidase 5 (aox5; formerly, aox3) (Parichy et al., 2000; McMenamin et al., 2014), exhibited long, fast projections with distinctive, membraneous vesicles at their tips; vesicles were often left behind when the projection carrying them retracted or fragmented (Figure 1B,E; Video 1). Such projections were especially frequent at early stages of stripe formation (7–8 SSL; Figure 1C-left; Figure 1—figure supplement 1) (Parichy and Turner, 2003b; Parichy et al., 2009; Patterson and Parichy, 2013) and individual cells could extend several projections over 18 hr of time-lapse imaging (Figure 1C-right). Projections reached as far as 5–6 cell diameters and extended and retracted quickly (Figure 1D,E). Incidences of projection formation were not markedly altered in mutants lacking melanophores or iridophores, or in transgenic fish having supernumerary melanophores (Figure 1—figure supplement 2; Video 2). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Projections by cells of xanthophore lineage in zebrafish but not pearl. Left, in zebrafish D. rerio, an aox5+ cell in a mosaically labeled larva extends fast, thin cellular projections. Right, such projections are not apparent in two aox5+ cells in pearl. 10 min interval, 580 min total. https://doi.org/10.7554/eLife.12401.007 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Fast projections of aox5+ cells persist in backgrounds with altered numbers of melanophores or iridophores. Shown are representative fields illustrating fast projections (arrows) of aox5+ cells in the presence of excess melanophores (Kitlga++), an absence of melanophores (mitfa) and an absence of iridophores (ltk). 5 min interval, 375 min total. https://doi.org/10.7554/eLife.12401.008 By contrast, fast projections were extended only rarely by aox5+ cells of pearl danio (Figure 1C,E; Figure 1—figure supplement 1 Video 1). Likewise, fast projections were rare among melanophores of both species, marked by expression of tyrosinase related protein 1b (tyrp1b; Figure 1—figure supplement 3, Video 3) and were not observed for iridophores, marked by purine nucleoside phosphorylase 4a (pnp4a; Video 4) (Lang et al., 2009; Curran et al., 2010; McMenamin et al., 2014). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Fast projections were only infrequently produced by cells of the melanophore lineage. Melanophores labeled with tyrp1b:membrane-mCherry. Most cellular processes were robust in size and relatively slow moving, although some could extend over long distances (yellow arrowhead). Only rarely were fast cellular projections extended (white arrow near top of frame). M, lightly melanized, differentiating adult melanophore in the prospective stripe region. EL, brownish embryonic/early larval melanophore persisting at the horizontal myoseptum in the prospective interstripe region. *, macrophage carrying mCherry+ debris. 5 min interval, 785 min total. https://doi.org/10.7554/eLife.12401.009 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Iridophores did not extend fast projections. Iridophores that have started to differentiate in the prospective interstripe (and in the vicinity of persisting EL melanophores), labeled with membrane-GFP driven by the promoter of purine nucleoside phosphorylase 4a (pnp4a). Thin, short and relatively straight projections are often extended at cell edges (e.g., arrow). An individual pnp4a+ cell is observed dispersing from the mat of aggregated pnp4+ iridophores (arrowhead). 5 min interval, 475 min total. https://doi.org/10.7554/eLife.12401.010 Thus, long, fast, and vesicle-containing projections were produced exuberantly by cells of the zebrafish xanthophore lineage during stages of stripe formation, but were not common to other pigment cell classes in this species or to cells of the xanthophore lineage in pearl danio. Fast pigment cell projections are distinct in cytoskeleton and morphology from long filopodia We asked whether fast projections of pigment cells were similar to previously described cellular projections. Fast projections were considerably longer than typical (<10 µm) filopodia, that lacked vesicles (e.g., Figure 1—figure supplement 3D; and below). Longer, often relatively straight, actin-based filopodia, or ‘cytonemes,’ function in intercellular communication in other systems (Miller et al., 1995; Ramirez-Weber and Kornberg, 1999; De Joussineau et al., 2003; Cohen et al., 2010; Caneparo et al., 2011; Danilchik et al., 2013; Massarwa and Niswander, 2013; Sanders et al., 2013; Gradilla et al., 2014; Luz et al., 2014; Roy et al., 2014; Stanganello et al., 2015). Similar to cytonemes, fast pigment cell projections contained F-actin, as evidenced by labeling with fluorescent reporters fused to the calponin homology domain of utrophin (UtrCH) as well as LifeAct (Burkel et al., 2007; Riedl et al., 2008; Sanders et al., 2013) (Figure 2A,B). Figure 2 Download asset Open asset Fast projections harbor microfilaments and microtubules. (A, B) aox5+ projections and puncta (arrowheads) labeled for F-actin, revealed by UtrCH-mCherry (A) and LifeAct-mKate (B). (C) Projections contained tubulin, as revealed by Tuba1b-mCherry. In some instances tubulin was absent from vesicles (arrowhead, upper) and in other instances was found in vesicles (arrow, lower). (D) Accumulations of microtubule end binding protein EB3 fused to GFP (arrowhead), were present in vesicles as well (arrow); aox5 here drives mCherry. Scale bars: 20 µm (A); 10 µm (B, C, D). https://doi.org/10.7554/eLife.12401.011 In contrast to cytonemes, however, fast projections had highly meandering trajectories and the vesicles associated with them had diameters of 1.7 ± 0.1 µm (mean ± SE, n=22), considerably larger than the 30–200 nm exosome-like particles trafficked within cytonemes (Bischoff et al., 2013; Sanders et al., 2013; Gradilla et al., 2014). Also unlike cytonemes, filaments of fast projections contained tubulin, as indicated by localization of an alpha tubulin fusion protein; such labeling was present in some but not all projection-associated vesicles (Figure 2C). Suggesting active microtubule assembly, fast projections and vesicles also exhibited transient accumulations of microtubule plus-end binding protein EB3 (Figure 2D; Video 5). Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Microtubule plus-end binding protein EB3 localized transiently in airinemes. Upper, membrane labeling with aox5:membrane-mCherry. Middle, EB3-GFP fusion protein driven by aox5 promoter. Lower, merge. EB3-GFP is present transiently as airinemes extend (arrow). Green spots in background are accumulations of xanthophore pigment in neighboring cells. 5 min interval, 190 min total. https://doi.org/10.7554/eLife.12401.012 Fast projections were also 1–2 orders of magnitude longer than recently observed actin- and tubulin-containing projections of chick somitic epithelial cells (Sagar et al., 2015) and Drosophila germ line stem cells (Inaba et al., 2015), as well as cellular bridges that can transfer cytoplasmic materials between embryonic neural crest cells (McKinney et al., 2011). Finally, these projections differed markedly from long but slow and robust, pseudopodial-like processes sometimes extended by melanophores transiently during development (Video 3) and present in adult fish (Hamada et al., 2014). Given their distinctive morphology and cytoskeletal composition, as well as their function (below), we termed these fast projections ‘airinemes,’ for Iris, who—fleet as the wind on golden wings—delivered messages for the gods (Homer, ~8th cent. BCE), as well as for Sir George Biddell Airy, who described limits on optical resolution (Cox, 2012). Airinemes are produced by xanthoblasts within melanophore stripes, rather than xanthophores within interstripes As a first step towards understanding potential roles for airinemes in stripe formation, we sought to further characterize the cells that extend them. In zebrafish, aox5+ cells occur in the prospective interstripe, where they differentiate as xanthophores [beginning ~6.5 SSL (Parichy et al., 2009; Patterson and Parichy, 2013)]. Yet, aox5+ cells also occur at lower densities in developing and completed stripes, where they remain unpigmented or lightly pigmented (Figure 3A); we refer to these incompletely differentiated cells as xanthoblasts (McMenamin et al., 2014). Figure 3 Download asset Open asset Airinemes were produced by xanthoblasts within prospective stripe regions. (A) Detail of developing pattern in zebrafish, illustrating pigmented aox5+ xanthophores of the interstripe as well as unpigmented aox5+ xanthoblasts of the prospective stripe (insets, boxed regions shown at higher magnification). Larva shown is 8.6 SSL, when xanthophore pigment is more readily visible, but after the peak of airineme production (Figure 1C). (B) aox5+ cells in prospective stripe regions were more likely to extend airinemes than aox5+ cells of the interstripe (χ2=28.6, d.f.=1, p<0.0001, N=295 cells). (C) Xanthoblasts (upper) had numerous membrane blebs (arrowhead), whereas xanthophores (lower) had smooth surfaces and more lobular edges (7.8 SSL). (D) Forced differentiation (TH++) reduced the incidence of cells that extended airinemes (left; χ2=12.2, d.f.=1, p<0.0001, N=123 cells) and the numbers of airinemes extended by each cell (right; TH++; χ2=12.0, d.f.=1, p<0.05), whereas differentiation-arrest increased airineme production (TH–; χ2=29.6, d.f.=1, p<0.0001; 7.5 SSL) Scale bars: 50 µm (A); 10 µm (C). https://doi.org/10.7554/eLife.12401.013 To determine if interstripe and stripe populations of aox5+ cells differ in airineme production we compared behaviors of cells in these locations during the peak of airineme deployment (7.5 SSL; Figure 1C). We found that cells within stripe regions, presumptive xanthoblasts, were more likely to extend airinemes than were cells within the developing interstripe, presumptive xanthophores (Figure 3B). Inspection of aox5+ cells at high resolution further revealed that unpigmented xanthoblasts, but not pigmented xanthophores, had membrane blebs from which airinemes originated (Figure 3C; Videos 6, 7). Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Membrane blebs of xanthoblast. Shown is a static image of a single aox5+ xanthoblast within the prospective stripe region, illustrating numerous membrane blebs, limited primarily to the superficial (epidermal-facing) surface. https://doi.org/10.7554/eLife.12401.014 Video 7 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Airinemes arose from membrane blebs of xanthoblasts. Shown are corresponding views of same field exposed to highlight membrane blebs from which airineme originates (upper, red arrow) and airineme filament (lower). An additional bleb formed near the end of observation (yellow arrow, upper). 5 min interval, 300 min total. https://doi.org/10.7554/eLife.12401.015 To test experimentally if airineme production was contingent upon differentiation state, we exploited the thyroid hormone (TH) dependence of xanthophore differentiation: aox5+ cells are differentiation-arrested in fish lacking TH, but differentiate fully across the flank in fish expressing excess TH (McMenamin et al., 2014). We found that aox5+ cells arrested as xanthoblasts (TH–) produced more airinemes than aox5+ cells forced to differentiate as xanthophores (TH++; Figure 3D; Video 8). These findings are consistent with the interpretation that airineme production is differentiation-state specific within the xanthophore lineage (and see below) Video 8 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Airineme production was differentiation-state dependent. Left, a wild-type control aox5+ cell extended several airinemes. Middle, exuberant airineme production by differentiation-arrested aox5+ cells in hypothyroid (TH–) fish in which the thyroid had been ablated transgenically at 4 days post-fertilization (McMenamin et al., 2014). Right, forced aox5+ cell differentiation in hyperthyroid (TH++) mutant (opallus, tshrD632Y) resulted in failure of airineme production. Spots of autofluorescence are evident in neighboring cells lacking aox5:membrane-GFP. 5 min interval, 795 min total. https://doi.org/10.7554/eLife.12401.016 Airinemes are target-specific and required for melanophore consolidation into stripes To assess the significance of airinemes for pigment pattern formation we sought to block their production. Consistent with actin and tubulin cytoskeletal dependencies, airineme production was curtailed by acute treatment with the myosin II inhibitor blebbistatin (Kovacs et al., 2004) and the microtubule polymerization inhibitor nocodazole (Figure 4—figure supplement 1A), although longer term whole-fish treatments were lethal. By contrast, low levels of the Cdc42 small GTPase inhibitor ML141 (Surviladze et al., 2010) inhibited airineme production (Video 9) while allowing for extended whole-fish treatments. In larvae treated with ML141 through adult pigment pattern formation, melanophores occurred ectopically within the interstripe (Figure 4—figure supplement 1A,B). Video 9 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Pharmacological inhibition of airineme production. Representative fields in which fast projections are extended by aox5+ cells in DMSO-treated controls, but not during acute administration of nocodazole, ML141 or blebbistatin. Note that cells continue to change shape and extend some slow moving processes, similar to controls. Stationary fluorescent puncta are pigment autofluorescence in neighboring cells lacking aox5:membrane-GFP. 5 min interval, 450 min total. https://doi.org/10.7554/eLife.12401.017 To block airineme production specifically in the xanthophore lineage we constructed a TetGBD (‘Tet’) transgene (Knopf et al., 2010; Patterson and Parichy, 2013) driven by aox5 to express dominant negative Cdc42N17 (dnCdc42) (Kieserman and Wallingford, 2009), inducible in a temporally specific manner with dexamethasone and doxycycline (dd; Figure 4—figure supplement 2A). Cdc42 has roles in cytoskeletal organization and a variety of cell behaviors and its inhibition blocks filopodial extension in other systems (Etienne-Manneville, 2004; Wu et al., 2008; Chen et al., 2012; Sadok and Marshall, 2014; Stankiewicz and Linseman, 2014). We found that extended, low-level induction of dnCdc42 in aox5+ cells inhibited airineme production (Figure 4A; Video 10) without significantly affecting the production of short filopodia, lamellipodia-like protrusive activities, the numbers of differentiated xanthophores or melanophores, or the distributions of aox5+ cells (Figure 4—figure supplement 2B–F). Yet, in aox5:Tet:dnCdc42 fish inhibited for airineme production, melanophores persisted ectopically in the interstripe (Figure 4B), as for whole fish treated with ML141. Figure 4 with 4 supplements see all Download asset Open asset Airineme dependent patterning and airineme targeting specificities. (A) dnCdc42 blocks airineme extension (χ2=16.4, d.f.=1, p<0.0001, N=43 cells total). (B) Interstripe melanophores persisted when airinemes were blocked with dnCdc42. Cell states are indicated by logos in lower left corners (X, aox5+ xanthophore lineage). Insets, brownish melanophores persisting from embryonic/early larval pattern and gray–black adult melanophores. (C) Airinemes contacting melanophores (arrows; Me, early larval; Mn, new; Mp, previously differentiated; xan, xanthophore). (D) Stabilization times (median ± IQR) of aox5+ airinemes on cells of melanophore (M) or xanthophore (X) lineages for zebrafish, cx41.8 mutant zebrafish, and pearl danio. aox5+ airinemes of wild-type zebrafish were less likely to stabilize, and stabilized more briefly (*, both p<0.0001) after contacting cells of the xanthophore lineage as compared to melanophores; this target specificity was altered in cx41.8 mutant zebrafish as well as pearl danio. Y-axis is split for clarity. (E) Zebrafish aox5+ airinemes were most likely to stabilize on Me and Mn (*, p<0.0001; median ± IQR). (F) cx41.8 mutant airinemes stabilized on aox5+ cells (arrow). (G) Vesicle transfer (arrow) to melanophore. Scale bars: 200 µm (B); 50 µm (C); 50 µm (F); 25 µm (G). https://doi.org/10.7554/eLife.12401.018 Video 10 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Airineme production was inhibited by dnCdc42. Upon treatment with dd, non-transgenic control cells extended airinemes normally, whereas cells in fish transgenic for aox5:Tet:dnCdc42 failed to extend airinemes despite forming other processes. Stationary fluorescent puncta are pigment autofluorescence in GFP– cells. 5 min interval, 415 min total. https://doi.org/10.7554/eLife.12401.023 These findings suggested that airinemes promote the consolidation of melanophores into stripes. During normal stripe development embryonic melanophores persisting from the early larval pigment pattern occur near the horizontal myoseptum, within the prospective interstripe. Many of these cells move short distances to join the developing stripes (Parichy et al., 2000; Parichy and Turner, 2003b; Takahashi and Kondo, 2008; Patterson and Parichy, 2013; Patterson et al., 2014), and their brownish color makes them distinguishable from gray–black adult melanophores that differentiate post-embryonically (Quigley et al., 2004; Parichy and Spiewak, 2015). In wild-type controls, these brownish embryonic melanophores had translocated to the edges of adult stripes, whereas in aox5:Tet:dnCdc42-expressing fish these melanophores remained in the interstripe (insets, Figure 4B). Given these observations, we predicted th

Changes in gene activity are essential for evolutionary diversification. Yet, elucidating the cellular behaviors that underlie modifications to adult form remains a profound challenge. We use neural crest-derived adult pigmentation of zebrafish and pearl danio to uncover cellular bases for alternative pattern states. We show that stripes in zebrafish require a novel class of thin, fast cellular projection to promote Delta-Notch signaling over long distances from cells of the xanthophore lineage to melanophores. Projections depended on microfilaments and microtubules, exhibited meandering trajectories, and stabilized on target cells to which they delivered membraneous vesicles. By contrast, the uniformly patterned pearl danio lacked such projections, concomitant with Colony stimulating factor 1-dependent changes in xanthophore differentiation that likely curtail signaling available to melanophores. Our study reveals a novel mechanism of cellular communication, roles for differentiation state heterogeneity in pigment cell interactions, and an unanticipated morphogenetic behavior contributing to a striking difference in adult form.

Pigment patterns are useful for elucidating fundamental mechanisms of pattern formation and how these mechanisms evolve. In zebrafish, several pigment cell classes interact to generate stripes, yet the developmental requirements and origins of these cells remain poorly understood. Using zebrafish and a related species, we identified roles for thyroid hormone (TH) in pigment cell development and patterning, and in postembryonic development more generally. We show that adult pigment cells arise from distinct lineages having distinct requirements for TH and that differential TH dependence can evolve within lineages. Our findings demonstrate critical functions for TH in determining pigment pattern phenotype and highlight the potential for evolutionary diversification at the intersection of developmental and endocrine mechanisms.

BACKGROUND:A critical event in neural tube closure is the formation of median hinge points (MHPs) and dorsolateral hinge points (DLHPs). Together, they buckle the ventral midline and elevate and juxtapose the neural folds for proper neural tube closure. Dynamic cell behaviors occur at hinge points (HPs), but their molecular regulation is largely unexplored. Bone morphogenetic proteins (BMPs) have been implicated in a variety of neural tube closure defects, although the underlying mechanisms are poorly understood.METHODS:In this study, we used in vivo electroporations, high-resolution microscopy, and biochemical analyses to explore the role of BMP signaling in chick midbrain neural tube closure.RESULTS:We identified a cell-cycle-dependent BMP gradient in the midbrain neural plate, which results in low-level BMP activity at the MHP. We show that although BMP signaling does not have a role in midbrain cell-fate specification, its attenuation is necessary and sufficient for MHP formation and midbrain closure. BMP blockade induces MHP formation by regulating apical constriction and basal nuclear migration. Furthermore, BMP signaling is critically important for maintaining epithelial organization by biochemically interacting with apicobasal polarity proteins (e.g., PAR3). As a result, prolonged BMP blockade disrupts apical junctions, desegregating the apical (PAR3(+), ZO1(+)) and basolateral (LGL(+)) compartments. Direct apical LGL-GFP misexpression in turn is sufficient to induce ectopic HPs.CONCLUSIONS:BMPs have a critical role in maintaining epithelial organization, a role that is conserved across species and tissue types. Its cell-cycle-dependent modulation in the neural plate dynamically regulates apicobasal polarity and helps to bend, shape, and close the neural tube.