Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation

Melanophore Migration and Survival during Zebrafish Adult Pigment Stripe Development Require the Immunoglobulin Superfamily Adhesion Molecule Igsf11

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation

<i>igsf11</i> is expressed by pigment cells and their precursors.

<p>(A,B) In situ hybridization for <i>igsf11</i> transcript during the larval-to-adult transformation, showing an <i>igfs11</i>-expressing cell near the hypodermis (A, and higher magnification in B). e, epidermis; m, myotome. (C) Similar location to that shown in (B), illustrating a cell within the hypodermis (arrowhead), coexpressing Igsf11 cell (magenta) and mitfa:GFP (green). Nuclei in all immunofluorescence images are counterstained with DAPI (blue). (D,D′) A melanophore isolated in vitro expresses Igsf11 (red). (E). Extra-hypodermal Igsf11+ cells (arrowhead) also coexpressed Igsf11 (magenta) and mitfa:GFP, though some mitfa:GFP+ cells were Igsf11− (lower left of panels). Shown here are cells just ventral to the aorta (a). (F) RT-PCR showed that cell populations isolated by differential centrifugation and highly enriched for melanophores (mel) and xanthophores (xan) express <i>igsf11</i> transcript. <i>dct</i>, <i>dopachrome tautomerase</i>, expressed by melanophores; <i>aox3</i>, <i>aldehyde oxidase 3</i>, expressed by xanthophores. β-actin, loading control. (G) <i>igsf11</i> expression was detected in several additional tissue types dissected from adult fish. Scale bars: in (A) 40 µm for (A); in (B) 10 µm for (B); in (C) 10 µm for (C,E); in (D) 20 µm for (D).</p

<i>seurat</i> is required autonomously to the melanophore lineage.

<p>(A, B) Wild-type <i>Tg(bactin:GFP)</i> cells transplanted to <i>seurat</i> mutant hosts. Fish shown are juveniles (∼13 mm standardized standard length, SSL <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002899#pgen.1002899-Parichy3" target="_blank">[11]</a>) and were treated just prior to imaging with epinephrine, which contracts melanosomes towards cell bodies, thereby facilitating the detection of GFP fluorescence. (A) Chimeras that developed wild-type melanophores exhibited patches of restored stripes (<i>n</i> = 6). (A′) Detail of boxed region in A, showing GFP+ melanophores (e.g., arrow), as well as occasional GFP−, presumptive <i>seurat</i> mutant melanophores (e.g., arrowhead). (B) Chimeras in which wild-type melanophores failed to develop exhibited a <i>seurat</i> mutant pattern of dispersed melanophores (arrowheads; <i>n</i>>100). In the example shown here, wild-type GFP+ cells developed as epidermis (B′; shown at same magnification as B). (C) When wild-type melanophores differentiated in a <i>nacre</i> mutant background, patches of normal stripes developed (<i>n</i> = 3; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002899#pgen.1002899-Budi1" target="_blank">[16]</a>). (D) By contrast, when <i>seurat</i> mutant melanophores differentiated in <i>nacre</i> hosts, these cells retained a dispersed pattern, as in the <i>seurat</i> mutant (<i>n</i> = 8), indicating a failure of xanthophores, iridophores, or other cell types to rescue melanophore stripe organization. In additional experiments, in which <i>nacre; Tg(bactin:GFP)</i> cells were transplanted to <i>seurat</i> mutant hosts, the differentiation of <i>nacre-</i> GFP+ (<i>seurat</i>+) iridophores likewise failed to rescue melanophore stripes in the <i>seurat</i> mutant background (donor xanthophores did not develop in these chimeras; data not shown). Scale bars: in (A) 100 µm for (A,B,B′); in (A′) 20 µm for (A′); in (C) 500 µm for (C,D).</p

Melanophore precursors require <i>igsf11</i> for their migration and survival.

<p>(A) Selected frames from time-lapse movies of mitfa:GFP+ cells in wild-type and seurat mutant explants. A single cell (red arrow) moved from dorsal to ventral over the duration of the movie. In a <i>seurat</i> mutant, many cells failed to migrate (e.g., red arrow) or died (yellow arrow) during the period of imaging. (B) Velocities (mean±SE) of mitfa:GFP+ cells were significantly reduced in <i>seurat</i> mutants compared to the wild-type (<i>t</i> = 11.2, d.f. = 135), as were total distances traveled (not shown). (C) <i>seurat</i> mutant melanophores were also significantly more likely to die than were wild-type melanophores (<i>X²</i> = 29.8, d.f. = 1).</p

Defective adult pigment stripes in <i>seurat</i> mutants.

<p>Shown are adult fish (A, B, C) and details of patterns (A′, B′, C′). (A, A′) Wild-type fish exhibit several dark stripes of melanophores and iridophores, as well as light “interstripes” of xanthophores and iridophores. (B, B′) Homozygous <i>seurat^utr15e1</i> mutants exhibit irregular spots of melanophores. (C, C′) <i>seurat^wp15e2</i>/<i>seurat^utr15e1</i> fish exhibit a less severe stripe defect, most evident ventrally (arrowhead). <i>seurat^wp15e3</i>/<i>seurat^utr15e1</i> exhibit a phenotype indistinguishable from <i>seurat^wp15e2</i>/<i>seurat^utr15e1</i>. At this stage, <i>seurat^wp15e2</i> and <i>seurat^wp15e3</i> are nearly indistinguishable from the wild-type when homozygous, though phenotypes are more apparent during the initial stages of stripe formation (not shown). Scale bar: in (A′), 1 mm for (A′–C′).</p

<i>igsf11</i>-dependent migration and survival of melanophores.

<p>(A) Repeated images of developing wild-type and <i>seurat</i> mutant larvae between 14–28 days post-fertilization. Numbers to the left of images are SSL. In wild-type larvae, new adult melanophores differentiated already within stripes or translocated short distances as stripes formed (e.g., note changes in the relative positions of cells 2 vs. 4, and cell 3 vs. 1 and 5). In <i>seurat</i> mutants, however, little movement was observed and many melanophores died as evidenced by the presence of melanized cellular debris apparent at high magnification (not shown; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002899#pgen.1002899-Parichy7" target="_blank">[21]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002899#pgen.1002899-Lang1" target="_blank">[32]</a>. Images shown were rescaled to maintain the same field of view as the fish grew; scale bars at 7.2 SSL and 11.2 SSL represent 100 and 200 µm, respectively. (B) When cultured <i>in vitro</i>, wild-type melanophores migrated further than <i>seurat</i> mutant melanophores. Shown are tracks of 16 cells of each genotype. (C) Quantification of total and rectilinear distances moved by cells <i>in vitro</i> confirmed reduced motility of <i>seurat</i> mutant melanophores (<i>t</i> = 3.0, <i>t</i> = 5.4, respectively; d.f. = 26). Shown are means ± SE.</p

Igsf11 promotes aggregation of K562 myeloid leukemia cells in vitro.

<p>Cells were transfected with wild-type zebrafish <i>igsf11</i> (A, B), S151P, <i>seurat^utr15e1</i> mutant <i>igsf11</i> (C, D), T29P, <i>seurat^wp15e2</i> mutant <i>igsf11</i> (E, F), or mock transfected (G, H). At the start of the experiment, the numbers of small cellular aggregates and total numbers of cells were similar (A, C, E, G). By 120 min, a relatively small number of aggregates containing numerous cells had formed in the cells transfected with wild-type <i>igsf11</i> (arrowheads in B), though this was not the case in cells of other treatments (D, F, H). (I) Quantitation of the ratio of cellular aggregates to the total number of cells (mean±SE) confirmed that cells were increasingly found in fewer, larger aggregates when transfected with wild-type <i>igsf11</i>. At 120 min, degrees of aggregation differed significantly among treatments overall (<i>F</i>_3,48 = 19.8, <i>P</i><0.0001). <i>Post hoc</i> means comparisons indicated that aggregation behavior in cells transfected with wild-type <i>igsf11</i> (B in the figure) differed significantly from that of cells transfected with mutant <i>igsf11</i> or controls (A in the figure; Tukey Kramer post hoc comparisons, <i>P</i><0.01); aggregation of cells transfected with mutant forms of <i>igsf11</i> did not differ significantly from one another or from mock transfected cells. Values shown are least squares means adjusted to control for variation after controlling for minor but significant variation among replicates (<i>P</i><0.01).</p

<i>seurat</i> mutants exhibit lesions in <i>igsf11</i>.

<p>(A) Meiotic mapping of the <i>seurat^utr15e1</i> allele revealed a ∼210 kb critical genetic interval harboring several open reading frames of which only <i>igsf11</i> exhibits ENU-induced lesions. Differences in numbers of individuals tested across markers reflect the absence of polymorphisms in some mapping families. (B) Schematic of inferred Igsf11 protein showing identified mutations and predicted domains. The lesion in the mapped allele, <i>seurat^utr15e1</i>, occurred in exon 4, whereas lesions in <i>seurat^wp15e2</i> and <i>seurat^wp15e3</i> were found in exon 2. S, predicted signal sequence; TM, predicted transmembrane domain; Ig (V-set), immunoglobulin V-set domain; Ig (I-set), immunoglobulin I-set domain.</p