1. Developmental Biology
  2. Evolutionary Biology
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Long-distance communication by specialized cellular projections during pigment pattern development and evolution

  1. Dae Seok Eom
  2. Emily J Bain
  3. Larissa B Patterson
  4. Megan E Grout
  5. David M Parichy  Is a corresponding author
  1. University of Washington, United States
Research Article
Cite this article as: eLife 2015;4:e12401 doi: 10.7554/eLife.12401
6 figures and 19 videos


Figure 1 with 3 supplements
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).

Figure 1—figure supplement 1
Developing adult pigment patterns during peak stages of aox5+ fast projections in zebrafish.

In zebrafish (7.5 SSL), melanophores are found along the horizontal myoseptum (e.g., outline and inset) and an interstripe region with pigmented xanthophores (inset, arrow) has started to develop. Vertical bars at left indicate approximate locations of stripes (black) and interstripes (orange). In pearl, fewer melanophores are present, the developing interstripe is indistinct and xanthophores have differentiated widely across the flank. Scale bar: 200 µm.

Figure 1—figure supplement 2
Zebrafish aox5+ cells extend fast projections independently of melanophores and iridophores.

(A) aox5+ projections persisted in microphthalmia a (mitfa) mutants (Lister et al., 1999), lacking melanophores, though were somewhat less frequent than in wild-type (wt; χ2=7.4, d.f.=1, p<0.01). The incidence of aox5+ cells extending projections did not differ from wild-type in leucocyte tyrosine kinase (ltk) mutants (Lopes et al., 2008) that lack iridophores (p=0.1). (B) Left, excess interstripe melanophores were generated by repeated heat-shock induction of ubiquitous Kit ligand a (Kitlga) (Patterson and Parichy, 2013; Patterson et al., 2014) (F1,5=12.9, p<0.05; control, heat-shocked wild-type siblings lacking the transgene). Middle, Increased numbers of melanophores did not affect the frequency with which projections were extended by aox5+ cells (p=0.9). Right, Despite increased numbers of melanophores, there were no effects on interstripe xanthophore numbers (p=0.8), suggesting that aox5+ cells do not alter their differentiation in response to whether or not they are in the vicinity of melanophores.

Figure 1—figure supplement 3
Rare fast projections of zebrafish melanophores.

(A) Incidences of projections by tyrp1b+ and aox5+ cells of melanophore and xanthophore lineages, respectively. (B and C) Examples of tyrp1b+ melanophores extending projections that were indistinguishable from those of aox5+ cells; labeling: membrane targeted mCherry (B) and GFP (C). (D) High resolution image of tyrp1b+ melanophore, illustrating abundant, short filopodial projections (arrowhead). Scale bars: 50 µm (B and C); 10 µm (D).

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).

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).

Figure 4 with 4 supplements
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).

Figure 4—figure supplement 1
Pharmacological blockade of airineme production.

(A) Cytoskeletal inhibitors reduced projection formation relative to controls (overall: χ2=24.0, d.f.=3, p<0.0001; among inhibitors: χ2=1.7, d.f.=2, p=0.4, N=212 cells). (B) Treatment with Cdc42 inhibitor ML141 resulted in excess interstripe melanophores (arrow). Fish were treated with epinephrine to partially contract pigment granules toward cell centers. Scale bar: 200 µm (B).

Figure 4—figure supplement 2
Expression of dnCdc42 transgene for inhibition of airinemes.

(A) Trunk cross-section during pigment pattern formation (~7.5 SSL) for individual expressing aox5:Tet:nVenus-2a-dnCdc42 mosaically and stained for Venus immunoreactivity. Nuclear-localizing Venus was co-expressed with xanthophore lineage marker Pax7 (Minchin and Hughes, 2008) (arrow; arrowhead indicates a Pax7+ cell not carrying the transgene). (B) High-resolution micrograph of aox5+ xanthoblast in wild-type zebrafish (7.5 SSL) illustrating airineme with vesicle (arrow) as well as short filopodia that lack vesicles (e.g., arrowhead). (C) Frames from time-lapse imaging (5 min intervals) showing aox5+ cells after several days of dd treatment to express dnCdc42 at low levels. Short filopodia (e.g., arrowheads) and other protrusions (e.g., *) were extended despite inhibition of airineme production. (D) Analyses of protrusive activities in time-lapse videos revealed no significant differences in numbers of short filopodia extended (left: t58=1.07, p=0.3), lengths of lamellipodial protrusions (center: t54=1.20, p=0.2), or speeds of lamellipodial extension (right: t54=0.62, p=0.4). (E) Following extended dd-induction of dnCdc42 (6.5–12.5 SSL), there were not significant differences in the total numbers of xanthophores (t8=0.83, p=0.4) or melanophores (t8=0.27, p=0.8) relative to dd-treated non-transgenic controls. (F) Distributions of aox5+ cells were similar between dd-treated dnCdc42 and control larvae. Scale bars: 20 µm (A); 5 µm (C); 20 µm (C); 50 µm (F).

Figure 4—figure supplement 3
Prolonged airineme contact with motile melanophores.

(A) Example of aox5+ airineme (arrow) contacting tyrp1b+ melanophore (yellow 1), with vesicle (arrowhead) that persisted at least 90 min; a second melanophore is adjacent (red 2) (B) Contacted melanophores were motile: over several days, melanophore 1 traversed melanophore 2 and came to rest in a relatively more dorsal position within the developing stripe. hm, horizontal myoseptum. xan, xanthophore. Scale bars: 20 µm (A); 20 µm (B).

Figure 4—figure supplement 4
Connexin dependence of airineme production.

(A) Phenotypes of wild-type and cx41.8 mutant zebrafish. (B) Stage-specificity of airineme production and numbers of airinemes produced per cell differed between wild-type and cx41.8 mutant zebrafish (left, genotype x stage interaction: χ2=94.2, d.f.=1, p<0.0001, N=639 cells; right, χ2=51.1, d.f.=7, p<0.0001, N=428 cells). Scale bar: 1 mm (A).

Figure 5 with 5 supplements
Melanophore clearance requires airineme-dependent Notch signaling in melanophores.

(A) aox5+ airineme vesicles harbored DlC-mCherry (arrow). (B) Frequencies of Tp1+ Notch-responding melanophores were reduced in the absence of aox5+ cells (csf1ra; χ2=15.5, d.f.=1, p<0.0001, N=77 cells) or airinemes (dnCdc42; χ2=9.0, d.f.=1, p<0.005, N=46 cells). Epidermal and muscle cells were also Tp1+ (not shown) but within the hypodermis, where pigment cells reside (Hirata et al., 2003), we observed only melanophores to be Tp1+. (C) Interstripe melanophores persisted when Notch signaling was blocked within the melanophore lineage by dnSu(H) [melanophore logo with green outlining], similar to dnCdc42 blockade of aox5+ airinemes (compare with Figure 4B). (D) Quantification of interstripe melanophores (mean ± SE) in dd-treated non-transgenic fish as well as mitfa:Tet:dnSu(H) and aox5:Tet:dnCdc42 (overall, F2,12=58.6, p<0.0001). (E) When induced with threshold levels of dd, persisting interstripe melanophores (means ± SE) were threefold more abundant (t7=2.9, p<0.05) in fish doubly transgenic for mitfa:Tet:dnSu(H) and aox5:Tet:dnCdc42 as compared to singly transgenic fish (shown at 9.8 SSL). Scale bars: 15 µm (A, left); 10 µm (A, right); 20 µm (B); 200 µm (C); 200 µm (E).

Figure 5—figure supplement 1
Delta-Notch gene expression and patterning consequences.

(A) Delta genes dlc and delta-like 4 (dll4) are expressed by isolated xanthophores of zebrafish and pearl danio. csf1r, xanthophore lineage marker; actb1, actin b1. notch1a is expressed by melanophores of both species. pmela, melanophore lineage marker. Fins containing numerous cell types are positive controls. Preliminary surveys also revealed a variety of additional Delta-Notch ligands and receptors expressed by cells in skin and other tissues in the environment of pigment cells that were not expressed detectably in xanthophores or melanophores (data not shown). (B) Zebrafish expressing constitutively active Notch intracellular domain (NICD) in the melanophore lineage exhibited wider stripes (t8=5.4, p<0.001) yet ~30% fewer melanophores (t8=6.9, p<0.0001) than non-transgenic siblings. Dashed yellow lines, approximate dorsal- and ventral-most stripe boundaries. (C) Zebrafish dlc mutants have more interstripe melanophores than wild-type siblings (t9=4.8, p<0.001), though total melanophore numbers were not different (P=0.6). Dashed lines, approximate location boundaries of interstripe as determined for wild-type fish (see Materials and methods). Scale bar: 200 µm (in B, for A and B).

Figure 5—figure supplement 2
Failure of stripe consolidation in xanthophore-deficient zebrafish.

Wild-type compared to csf1ra (panther) mutant zebrafish that lacks virtually all xanthophores (arrows) (Parichy et al., 2000; Patterson and Parichy, 2013). Melanophores were widely dispersed in the csf1ra mutants. Scale bars: 500 µm (upper); 100 µm (lower).

Figure 5—figure supplement 3
Notch signaling inhibition.

(A) Treatment with Notch inhibitor LY411575 during adult pigment pattern development caused defects in melanophore clearance and stripe consolidation. (B) Induction of mitfa:Tet:nVenus-2a-dnSu(H) in melanophores of stable transgenics reduced expression of Notch target gene her6 (p<0.05) in isolated melanophores, relative to non-transgenic but dd-treated controls. (C) Melanophore of fish mosaically expressing mitfa:Tet:nVenus-2a-dnSu(H) stained for nuclear-localizing Venus by immunohistochemistry (arrow); nucleus is bipartite. Scale bar. (A) Extended induction of mitfa:Tet:nVenus-2a-dnSu(H) in melanophores did not affect total numbers of xanthophores (t8=0.92, p=0.4) or melanophores (t8=0.70, p=0.5) relative to dd-treated non-transgenic controls. Fish analyzed here were performed concurrently with those of Figure 4—figure supplement 2E and employed the same controls. Scale bars: 200 µm (A); 20 µm (C).

Figure 5—figure supplement 4
Airinemes and patterning in the zebrafish caudal fin.

(A) aox5+ cell extending airineme (arrowhead) in the regenerating fin. (B) Regenerative fin regions examined. Red bar, amputation plane. From distal to proximal, pattern formation is on-going (‘intermingled’), melanophores are consolidating into stripes ('transitional'), or stripes and interstripes have been fully re-formed (‘completed’) (Rawls and Johnson, 2000). (C) Airinemes were produced most often by aox5+ cells of the transitional zone (χ2=17.4, d.f.=2, p<0.0005, N=651 cells), though were less frequently observed than during the adult body pattern development. (D) Wild type, controls treated with dd during fin ontogeny had melanophores principally within stripes (arrowhead) whereas dd-treated individuals stably expressing dnCdc42 or dnSu(H) in the xanthophore or melanophore lineages, respectively, had melanophores that were more widely dispersed (arrows). Scale bars: 25 µm (A); 2 mm (B); 100 µm (C).

Figure 5—figure supplement 5
Dependence of melanophore kita expression on airinemes and Notch signaling.

(A) kita heterozygosity sensitized melanophores (means ± SE) for reduced Notch signaling [dnSu(H); t7=4.0, p<0.005] or airineme production (dnCdc42; t6=4.0, p<0.005). (B) Excess interstripe melanophores in kita heterozygotes compared to fish homozygous wild type for kita when Notch signaling or airinemes are inhibited. (C) kita expression (means ± SE) was reduced upon depletion of xanthophore lineage, Notch signaling or airinemes (one-tailed, paired t2=9.9, 7.6, 5.0; all p<0.05). (D) In pearl melanophores, Notch signaling (her6; Figure 5—figure supplement 1C) and kita expression (means ± SE) were reduced in comparison to zebrafish melanophores (one-tailed, paired t2=3.4, 3.4; p<0.05). Scale: 200 µm (B).

Figure 6 with 1 supplement
Factors extrinsic to the aox5+ cells inhibit airineme production and signaling in pearl and models for airineme signaling and its evolution.

(A) Left, Pearl aox5+ cells extended more airinemes when differentiation-arrested (TH–; χ2=12.5, d.f.=1, p<0.0005, N=412 cells). Right, zebrafish aox5+ cells transplanted to pearl, or receiving excess Csf1 in zebrafish, extended fewer airinemes than comparably staged aox5+ cells in unmanipulated zebrafish (χ2=23.1, 22.1, d.f.=1, 1; p<0.0001, N=846 cells total). (B) In chimeras resulting from transplants of zebrafish donors (aox5:membrane-GFP, ubiquitous ubb:mCherry [Mosimann et al., 2011]) to pearl hosts, zebrafish aox5+ cells (arrow) were typically intermingled with pearl melanophores, as well as ubb+ zebrafish melanophores (arrowheads). (C) Working models for pigment cell interactions and pattern formation in zebrafish (left) and pearl danio (right). In zebrafish, xanthoblasts in stripe regions extend airinemes that signal to melanophores (Xb→M), promoting their clearance from the interstripe during stripe consolidation. Results of this study are consistent with interactions involving xanthoblast airineme dependent Delta (Dlc or possibly Dll4) activation of Notch signaling in melanophores, and the potentiation of Kita-dependent melanophore motility. Nevertheless, these data do not exclude roles for additional modes of Delta–Notch signaling, or the possibilities that airinemes transduce additional signals, or signals that cannot be distinguished from the Delta–Notch pathway using the experimental paradigms here employed. Analyses of mutant zebrafish further support roles for Cx41.8 in contributing to airineme-dependent communication, through modulation of target specificity or xanthophore lineage differentiation. In addition to xanthoblast–melanophore interactions, iridophores have attractive and repulsive effects on melanophores (I→M; I⊣M) (Frohnhofer et al., 2013; Patterson and Parichy, 2013) and express Csf1, promoting the differentiation of xanthophores (Xb→X) at the interstripe (Patterson and Parichy, 2013). Differentiated xanthophores repel melanophores (X⊣M) during normal development (Nakamasu et al., 2009) and are capable of repressing iridophore organization (X⊣I) [for details, see: (Patterson et al., 2014)]. In pearl danio, Csf1 is expressed at elevated levels by cells other than iridophores and this drives earlier and broader xanthophore differentiation than in zebrafish (Patterson et al., 2014). Precocious, widespread differentiation of xanthophores likely limits directional cues available to melanophores while simultaneously curtailing the potential for airineme signaling, as airineme competent xanthoblasts are depleted. Tissue contexts (lower panels) also show eventual death of some melanophores remaining in the interstripe in zebrafish (Parichy et al., 2000; Parichy and Turner, 2003b) and the higher overall incidence of melanophore death in pearl danio (Quigley et al., 2005); iridophores are omitted for clarity. Scale bar: 50 µm (B).

Figure 6—figure supplement 1
Interspecific chimeras reveal non-autonomous effects on pattern and aox5+ cell behaviors.

(A) In zebrafish→pearl chimeras, well-organized stripes of zebrafish cells formed but only when all three classes of zebrafish pigment cells (inset: x, xanthophore; m, melanophore; i, iridophores) were present and in the vicinity of other zebrafish tissues (blue outline, donor myotomes). Ten adult chimeras analyzed. (B) In pearl→albino zebrafish chimeras (in which host melanophores were present but unpigmented), pearl melanophores were confined to stripes, adopting a zebrafish-like arrangement; stripe-interstripe boundaries are indicated by dashed white lines and interstripes by yellow-orange bars at left. (C) Pearl aox5+ cells frequently developed in zebrafish hosts at embryonic/early larval stages (N=90 aox5+ chimeras), yet these same cells typically died by prior to time-lapse imaging during adult pigment pattern formation, here apparent by fragmentation and extrusion of GFP+ debris, typical of pigment cell death in zebrafish (Lang et al., 2009Parichy et al., 1999). (D) Prior to extrusion, rare, surviving pearl danio aox5+ cells could extend numerous airinemes in zebrafish hosts (3 airinemes are shown; *, aox5+ cell fragmenting during imaging). Scale bars: 100 µm (A, for A and B); 20 µm (C); 50 µm (D).



Video 1
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.

Video 2
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.

Video 3
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.

Video 4
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.

Video 5
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.

Video 6
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.

Video 7
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.

Video 8
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.

Video 9
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.

Video 10
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.

Video 11
Airinemes extended by cells of the xanthophore lineage stabilized on melanophores.

Shown are prospective dorsal stripe regions of two individuals; prospective interstripe is at bottom of frame. In each view, an aox5+ cell (green) extends an airineme (arrow) that stabilized (*) on a nearby tyrp1b:mCherry+ melanophore (magenta). EL, persisting embryonic/early larval melanophore. M, lightly pigmented adult melanophore. M*, more heavily pigmented adult melanophore. Video shows brightfield views before and after time-lapse. 5 min interval, 475 min total.

Video 12
Airineme targeting differed among melanophore subpopulations.

Several previously differentiated tyrp1b+ melanophores (M; magenta) are shown as well as an initially unpigmented tyrp1b+ melanoblast (mb, upper left). Although numerous airinemes were extended by aox5+ cells in the vicinity of melanophores, only one (yellow arrow) stabilized (*) on a previously differentiated melanophore. Another airineme stabilized on the differentiating melanoblast (yellow arrowhead), which received additional aox5+ puncta. Video hows brightfield views before and after time-lapse and illustrates acquisition of melanin by as the melanoblast continued to differentiate. 5 min interval, 905 min total.

Video 13
Airineme-delivered membraneous vesicles persisted on melanophores.

Left, aox5+ cell (green) extending airineme. Middle, tyrp1b+ melanophores (magenta). Right, merge. After the airineme contacted the melanophore it retracted, leaving behind a GFP+ vesicle that persisted on the melanophore membrane (arrow). 5 min interval, 310 min total.

Video 14
Membrane vesicles could be transferred in the apparent absence of airinemes.

Left, aox5+ cell (green). Middle, tyrp1b+ melanophores (magenta). Right, merge. Several vesicles are indicated by yellow and white arrows. 5 min interval, 325 min total.

Video 15
Altered airineme targeting altered by connexin mutation.

Upper, wild-type zebrafish airinemes (white arrow) retracted rapidly after contacting other aox5+ cells. Lower, leopard (cx41.8) mutant airinemes (red arrow) frequently stabilized on other aox5+ cells. 5 min interval, 450 min total.

Video 16
Rare airinemes projected by pearl aox5+ cells stabilized on xanthophores.

Representative airinemes (arrowheads) stabilized on xanthophores, marked by orange pigment granule at cell centers (e.g., x). 5 min interval, 385 min total.

Video 17
Airineme vesicles harbored Dlc-mCherry.

Left, aox5+ cell (green) extending airineme. Middle, dlc:Dlc-mCherry (magenta). Right, merge. 5 min interval, 170 min total.

Video 18
Zebrafish aox5+ cells transplanted to pearl hosts behaved like pearl aox5+ cells.

Zebrafish-derived cells expressed ubb:mCherry (magenta) and zebrafish cells of the xanthophore lineage were co-labeled with aox5:membrane-GFP (green). Airinemes were very rarely extended by zebrafish aox5+ cells in the pearl background; a single such example is shown (arrow). Also visible are ubb+ muscle cells deeper in the fish. Spots of green were autofluorescing host xanthophores, and melanophores shown in bright field were of host origin. 5 min interval, 370 min total.

Video 19
Transgenic expression of hsp70l:Csf1a blocked airineme production in zebrafish.

Upper, an aox5+ cell extends an airineme (arrow) in a heat-shocked, non-transgenic control fish, that stabilized on an adjacent melanophore (magenta). Additional aox5+ vesicles were evident as well. Lower, in heat-shocked transgenic fish in which differentiation had been forced by Csf1a overexpression, airineme and vesicle production were not evident. 5 min interval, 810 min total.


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