Post-Embryonic Nerve-Associated Precursors to Adult Pigment Cells: Genetic Requirements and Dynamics of Morphogenesis and Differentiation

The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia

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

Basonuclin-2 Requirements for Zebrafish Adult Pigment Pattern Development and Female Fertility

Relatively little is known about the generation of adult form. One complex adult trait that is particularly amenable to genetic and experimental analysis is the zebrafish pigment pattern, which undergoes extensive remodeling during post-embryonic development to form adult stripes. These stripes result from the arrangement of three classes of neural crest-derived pigment cells, or chromatophores: melanophores, xanthophores, and iridophores. Here, we analyze the zebrafish bonaparte mutant, which has a normal early pigment pattern but exhibits a severe disruption to the adult stripe pattern. We show that the bonaparte mutant phenotype arises from mutations in basonuclin-2 (bnc2), encoding a highly conserved, nuclear-localized zinc finger protein of unknown function. We show that bnc2 acts non-autonomously to the melanophore lineage and is expressed by hypodermal cells adjacent to chromatophores during adult pigment pattern formation. In bonaparte (bnc2) mutants, all three types of chromatophores differentiate but then are lost by extrusion through the skin. We further show that while bnc2 promotes the development of two genetically distinct populations of melanophores in the body stripes, chromatophores of the fins and scales remain unaffected in bonaparte mutants, though a requirement of fin chromatophores for bnc2 is revealed in the absence of kit and colony stimulating factor-1 receptor activity. Finally, we find that bonaparte (bnc2) mutants exhibit dysmorphic ovaries correlating with infertility and bnc2 is expressed in somatic ovarian cells, whereas the related gene, bnc1, is expressed within oocytes; and we find that both bnc2 and bnc1 are expressed abundantly within the central nervous system. These findings identify bnc2 as an important mediator of adult pigment pattern formation and identify bonaparte mutants as an animal model for dissecting bnc2 functions

A Model of Mindful Parenting: Implications for Parent–Child Relationships and Prevention Research

This paper introduces a model of “mindful parenting” as a framework whereby parents intentionally bring moment-to-moment awareness to the parent–child relationship. This is done by developing the qualities of listening with full attention when interacting with their children, cultivating emotional awareness and self-regulation in parenting, and bringing compassion and nonjudgmental acceptance to their parenting interactions. First, we briefly outline the theoretical and empirical literature on mindfulness and mindfulness-based interventions. Next, we present an operational definition of mindful parenting as an extension of mindfulness to the social context of parent–child relationships. We discuss the implications of mindful parenting for the quality of parent–child relationships, particularly across the transition to adolescence, and we review the literature on the application of mindfulness in parenting interventions. We close with a synopsis of our own efforts to integrate mindfulness-based intervention techniques and mindful parenting into a well-established, evidence-based family prevention program and our recommendations for future research on mindful parenting interventions

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Interactions with Iridophores and the Tissue Environment Required for Patterning Melanophores and Xanthophores during Zebrafish Adult Pigment Stripe Formation

<div><p>Skin pigment patterns of vertebrates are a classic system for understanding fundamental mechanisms of morphogenesis, differentiation, and pattern formation, and recent studies of zebrafish have started to elucidate the cellular interactions and molecular mechanisms underlying these processes. In this species, horizontal dark stripes of melanophores alternate with light interstripes of yellow or orange xanthophores and iridescent iridophores. We showed previously that the highly conserved zinc finger protein Basonuclin-2 (Bnc2) is required in the environment in which pigment cells reside to promote the development and maintenance of all three classes of pigment cells; <i>bnc2</i> mutants lack body stripes and interstripes. Previous studies also revealed that interactions between melanophores and xanthophores are necessary for organizing stripes and interstripes. Here we show that <i>bnc2</i> promotes melanophore and xanthophore development by regulating expression of the growth factors Kit ligand a (Kitlga) and Colony stimulating factor-1 (Csf1), respectively. Yet, we found that rescue of melanophores and xanthophores was insufficient for the recovery of stripes in the <i>bnc2</i> mutant. We therefore asked whether <i>bnc2</i>-dependent iridophores might contribute to stripe and interstripe patterning as well. We found that iridophores themselves express Csf1, and by ablating iridophores in wild-type and mutant backgrounds, we showed that iridophores contribute to organizing both melanophores and xanthophores during the development of stripes and interstripes. Our results reveal an important role for the cellular environment in promoting adult pigment pattern formation and identify new components of a pigment-cell autonomous pattern-generating system likely to have broad implications for understanding how pigment patterns develop and evolve.</p></div

Interstripe xanthophores developed after iridophores in wild-type larvae and were further delayed in <i>bnc2</i> mutants.

<p>Shown are a representative wild-type (<i>bnc2</i>/+) larva (A) and a sibling <i>bnc2</i> mutant (B) imaged repeatedly over 27 d beginning at 6.0 SSL, just prior to the appearance of iridophores at the anteroposterior region imaged, dorsal to the anus. In both the wild-type and the <i>bnc2</i> mutant iridophores started to appear by day 2 of imaging (blue arrowheads). Xanthophores started to differentiate by day 9 of imaging in wild-type; newly arising xanthophores are indicated by red dashed circles. In contrast, xanthophores did not appear until day 25 of imaging in the <i>bnc2</i> mutant. As iridophores (and xanthophores) in the interstripe became more abundant, some early larval melanophores along the horizontal myoseptum disappeared from view (e.g., green arrows in A, d12 and d15). For easier visualization of melanophores and other cell type, fish were treated briefly with epinephrine immediately prior to imaging, which contracts melanosomes towards the cell body; the distribution of melanin thus indicates the centers of melanophores whereas processes extending out from the cell body are not visible. Bottom panels schematize the distribution of iridophores (light blue) and xanthophores (red) on the final day shown. Samples sizes for which complete image series were obtained were: <i>bnc2</i>, <i>n</i> = 4; <i>bnc2/+</i>, <i>n</i> = 6. Scale bar: in (B, d27) 80 µm for (A,B).</p

Melanophore and xanthophore development is disrupted in additional iridophore-deficient mutants.

<p>(A) Comparison of xanthophore and melanophore development in wild-type and <i>ltk</i> mutants. Shown are details at the horizontal myoseptum from larger images of representative wild-type (<i>ltk</i>/+) and <i>ltk</i> mutant, stage-matched siblings imaged daily (beginning at 6 SSL). In the wild-type, nearly all melanophores persisted through the image series. A xanthophore had already developed at the onset of imaging (day 0, red dashed circle), and additional xanthophores differentiated shortly thereafter. In the <i>ltk</i> mutant, however, melanophores were frequently lost between days (green arrowheads) and melanin-containing debris and extrusion bodies were often apparent (green arrows). Unlike the wild-type, no xanthophores differentiated until day 5 of imaging. (B) During later development (9.6 SSL), xanthophores were confined principally to the interstripe region of the wild-type whereas xanthophore developed widely over the flank in the <i>ltk</i> mutant. The horizontal myoseptum lies at the lower edge of both images. Lower panels show positions of xanthophores in red. (C) Comparison of wild-type and <i>ednrb1a</i> mutant. Shown are ventral flanks of representative stage-matched, sibling wild-type (<i>ednrb1a</i>/+) and <i>ednrb1a</i> mutant larvae imaged daily (8.8–10 SSL). At the onset of imaging, wild-type melanophores are largely absent from a region where the second interstripe will form by day 7 of imaging (blue bars). In <i>ednrb1a</i> mutants, however, melanophores are relatively uniformly distributed in this region at the onset of imaging, and, by day 7 of imaging, formed clusters where the second interstripe would normally form (green bars). Images shown were rescaled to control for growth. (D) Closeups showing reduced iridophores in <i>ednrb1a</i> mutant compared to wild-type (9.0 SSL) as well as wider distribution of xanthophores. Fish in A, B and D were treated briefly with epinephrine prior to imaging. Sample sizes for which complete image series were obtained were: <i>ltk</i>, <i>n</i> = 6; <i>ltk/+</i>, <i>n</i> = 5; <i>ednrb1a</i>, <i>n</i> = 4; <i>ednrb1a/+</i>, <i>n</i> = 5. Scale bars: in (A, d0) 60 µm for (A); in (B) 100 µm for (B); in (C, d0) 200 µm for (C, d0); in (D) 100 µm for (D).</p