Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning

Zebrafisch

Introduction

Sequential and periodic structures formation is a fundamental issue in developmental biology. The skin pigment pattern of zebrafish is a good model system to study the mechanism of biological pattern formation. It is known that interactions between melanophores and xanthophores play a key role in the formation of adult pigment stripes, molecular mechanisms for these interactions remain largely unknown. Theory that explains various examples of biological pattern formation suggests that such patterns result from a stationary wave (Turing pattern or reaction-diffusion pattern) made by a combination of putative reactive and diffusible substances. With zebrafish there is three types of pigment cells: melanophores, xanthophores and iridophores [1]. Experiments suggested that pigment patterns result from interactions between these three types of pigment cells. Also, according to Turing’s theory, long-range interactions of melanophores and xanthophores are more important because they determine the width of stripes. The aim is to identify the molecular and cellular mechanism of long-range interaction.

Expression of Notch family members and their ligands in pigment cells

Any excess amount of signal protein should alter the resulting patterns. Hence, six candidate genese were expressed ectopically in the melanophore lineage using the mitfa promoter. For the initial screening, embryos were injected with each transgene, and reared at least ten injected fish to adulthood for each candidate gene. For deltaC, two of 15fish exhibited pattern alterations but other genes had no pattern differences. In situ hybridization was performed on late larval zebrafish and found that deltaC is expressed in presumptive xanthophores, but not in melanophores (Fig. 1A). Among the Notch family genes, notch1a was expressed abundantly in melanophores. Also, notch2 expression was detected relatively low in both melanophores and xanthophores (Fig. 1D). From these, it can be suggested that a Delta/Notch signal is transmitted from xanthophores (deltaC, delta-like 4 or both) to melanophores (notch1a, notch2 or both) to promote melanophore survival in zebrafish skin.

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Fig. 1. Expression analysis of Notch receptors and their ligands. (A,B) In situ hybridization for deltaC in larval fish. Presumptive xanthophores in the inter stripe are stained in wild type (A) but these cells and deltaC staining is absent in the csf1ra mutant (B).

Melanophore survival requires Notch signaling

This part of the experiment was done to determine whether melanophore survival depends on the Delta/Notch signal. As shown in Fig. 2A,F, treatment with DAPT for 15 days decreased the number of melanophores to 60% of that before the treatment. In DAPT-treated fish, melanin-containing debris were observed, a hallmark of melanophore death in the region from which melanophores had disappeared (Fig. 2D,E). However, after removing DAPT, melanophore numbers recovered quickly (Fig. 2F), suggesting that melanophore precursors were unperturbed. From this observation, to make sure that DAPT-induced death of melanophores reflected an inhibition of Notch signaling, stable transgenic lines expressing in the melanophore lineage either DeltaC or the Notch1a intracellular domain was generated. From this it showed that melanophores of Tg(mitfa:NICD1a) persisted even in the presence of DAPT, whereas melanophores of Tg fish were lost during DAPT treatment (Fig. 2B,C,F). It confirmed that melanophore death occurred specifically in response to Notch signaling inhibition.

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Fig. 2. Effect of Notch inhibitor on melanophores. (A-C) Altered melanophore distributions resulting from DAPT treatment over 15 days. (D) Death of melanophores. Two cells (circled by dotted lines) present at day 6 (left) were lost by day 9 (right), though residual melanin-containing debris remains visible. (E) Cross-section through the skin of a DAPT-treated fish. (F) Alteration of melanophore numbers during DAPT treatment, shown as the ratio of cells surviving at day X relative to the total number of cells at day 0.

Xanthophores are a source of the survival signal given to melanophores

As xanthophores express Notch ligands and elimination of xanthophores cause the death of melanophores, xanthophores are the likely source of the Notch signal. Comparison of the effects of transient xanthophore loss between wild-type and Tg fish. Laser ablation of xanthophores was done and 28% of melanophores died in wild-type fish (Fig. 4A), whereas no melanophores died in fish expressing NICD1a (Fig. 4B,C). Therefore, Notch pathway activation autonomous to melanophores can substitute for xanthophores, as the prediction was that xanthophores promote melanophore survival through Delta/Notch signaling.

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Fig. 4. Melanophore survival is independent of xanthophores in Tg(mitfa:NICD1a) transgenic fish. (A,B) Effects on melanophores after ablation of xanthophores is shown for wild type (A) and Tg(mitfa:NICD1a) (B). Red squares indicate the areas in which xanthophores were ablated. White circles indicate melanophores present on day 1 but lost by day 8. (C) Proportion of melanophores that died in wild-type and Tg(mitfa:NICD1a) fish.

Melanophores extend long projections to touch xanthophores directly

Delta and Notch belong to families of membrane-bound proteins. Hence, if a Delta/Notch survival signal is provided by xanthophores to melanophores, these cells must contact one another directly. As even melanophores distant from xanthophores died in response to xanthophore ablation, it was assumed that melanophores might contact xanthophores by cellular processed not normally apparent (Fig. 5A). Yohimbine (an alpha 2-adrenoceptor antagonist) was used to visualize thin cellular projections. From Fig. 5B, it shows that yohimbine caused the movement of melanosomes into long projections that extend towards xanthophores. Electron microscopy was done to further confirm that the melanophore projections directly touched xanthophores passing through the layer of iridophores.

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Fig. 5. Melanophores extend long projections. (A,B) Black stripe region of wild-type fish in the normal condition (A) and after addition of yohimbine (B), which reveals projections extending towards xanthophores. (C) Regions imaged in D-F. (D-F) Fluorescent images of melanophore membranes revealed by mitfa: EGFP-CAAX expression in  brass mutants. (D) Three melanophores in the black stripe extend long projections towards xanthophores. (E) A cluster of melanophores, each of which exhibits long projections. (F) Melanophores adjacent to a break in the stripe extend long projections radially.

Discussion

Xanthophores express Delta ligands (deltaC and delta-like 4), a family of membrane-bound ligands, and that melanophores express Notch receptors (notch1a and notch2). Next, Delta/Notch signaling promotes melanophore survival, and that this signal originates with xanthophores. Finally, melanophores in the black stripes extend long projections towards xanthophores that may enable pigment cells to transfer these long-range signals, despite association of Noth and Delta with the cell membrane was found. In future study, exploring that the Delta/Notch signal is transduced specifically at the sites of such projections, also to explain how the width of a stripe could be formed and stably maintained. Currently, these hypothesis is simple and useful to explain patterning, it remains to be confirmed by further experiments. This article presented the possibility that a long-range signal is likewise transmitted by direct contact between melanophores and distant xanthophores. There is, so far, no diffusible molecule demonstrated to be involved in pigment pattern formation.

Thoughts on Paper

This research has well explained the notch signaling that is done to the zebrafish. However it has been just tested to certain sites that during their future study how the width could be also stabilized while the stripes are being formed. The strength of this experiment is  knowing to separate between melanophores and xanthophores and distinguishing the difference they cause toward zebrafish patterning.

References

Hamada, H., Watanabe, M., Law, H. E., Nishida, T., Hasegawa, T., Parichy, D. M. and Kondo, S. (2014). Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning. Development. 141, 1418.

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Kopan, R. 2010. Notch Signaling. London: Academic Press.

Parichy, D. M. (2003). Pigment patterns: fish in stripes and spots. Curr. Biol. 13, R947-R950.

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