In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning

 

Background

The famous zebrafish stripes have often been used as a model for pigment patterning in animals. Three pigment cell types contribute to the stripe pattern – black melanophores in the stripes themselves, yellow xanthophores in the interstripe regions, and reflective iridophores which reside in both. Iridophores have different morphologies depending on their local environment – they are cuboidal and densely packed within interstripes, whereas within stripes they are more stellate and spaced out.

Previous work^[1] investigating how the zebrafish stripe pattern emerges and propagates has suggested that iridophores take a central role in organising the process. The current model is that iridophores proliferate, differentiate and pack together densely to form the first interstripe in juvenile fish. Over time, some of these interstripe iridophores adopt a looser morphology and migrate into developing stripes. After a period of proliferation, some of these ‘loose’ iridophores then reaggregate again to form a second interstripe. Therefore, a single iridophore cell type is predicted to transition between dense and loose configurations to pattern the fish.

However, this preprint by Gur, Bain and colleagues proposes an alternative model, whereby iridophore progenitors do not interconvert between dispersed and dense morphologies, but instead take cues from surrounding melanophores to differentiate into one of two iridophore subtypes – stripe and interstripe – with distinct physical and transcriptomic characteristics.

 

Key findings

In order to study the movements of iridophores during patterning, the team conducted live imaging experiments using the iridophore-specific fluorescent reporter line Tg(pnp4a:mCherry). Despite imaging larvae for over 300(!) hours, the authors did not observe any interconversion of pnp4a:mCherry+ iridophores between interstripes and stripes, conflicting with the previous migration model. To test this further, the team conducted a fate mapping experiment using a pnp4a:mCherry reporter line also containing nuclear mEos, a photoconvertible green to magenta fluorescent protein. A week after photoconversion of an interstripe region, pnp4a:mCherry cells had white nuclei, from a mixture of photoconverted magenta, and more recently translated green unconverted mEos fluorescence. In stripes however, only pnp4a:mCherry cells with green nuclei were observed, confirming that no interstripe iridophores migrated into this region.

This led the authors to hypothesise that rather than being from the same differentiated cell population, stripe and interstripe iridophores might represent distinct cell subtypes. To test this, they investigated the physical and transcriptional differences between the two in greater detail.

Iridophores are reflective because they contain stacks of crystalline guanine. Using cryogenic scanning electron microscopy and synchrotron-based micro X-ray diffraction, the authors showed that stripe iridophores contained regular, neatly-aligned guanine crystals. In contrast, guanine crystals in interstripe iridophores were highly disordered (see part of preprint Figure 3 below).

 

From preprint Figure 3 (with permission from authors). Left – blue stripe iridophores and black melanophores under incident illumination and under fluorescence after staining with malachite green. Underneath, cryo-SEM images show guanine crystal stack architecture. Right – as left, but for interstripe iridophores and yellow xanthophores.

 

So what might influence which iridophore subtype progenitors become? The team hypothesised that the surrounding environment, in particular stripe melanophores, may influence iridophore crystallotype. They compared the X-ray diffraction patterns in skin of mitfa genetic mutants, which completely lack melanophores, to albino mutants, in which melanophores are present but lack melanin. This revealed that the vast majority of iridophores were of the interstripe disordered crystallotype in mitfa mutant fish. However, in albino fish, ordered stripe iridophore crystallotypes were present within stripes of unpigmented melanophores, alternating with disordered interstripe crystallotypes. Additionally, they used a temperature sensitive allele of mitfa to conditionally add or remove melanophores, and observed that iridophores differentiated into the ordered crystallotype only when melanophores were present.

Taken together, this suggests that stripe and interstripe iridophores arise by in situ differentiation depending on their surrounding environment, and represent distinct cellular crystallotypes.

 

Why I chose this preprint

I must admit that in these unusual times I found the pretty images of zebrafish stripes in this preprint quite soothing! It was also good to learn more about iridophores, which aren’t nearly as well covered in the literature as melanophores for example. More specifically, I liked how the live imaging, fate mapping analyses, and different transgenic lines were used in conjunction with X-ray diffraction and SEM to investigate the guanine crystal structure – something I’ve not seen in zebrafish papers before but really neatly illustrates the differences between stripe and interstripe iridophores.

 

Questions for the authors

1. Previous work supporting the ‘morphogenic respecification’ model used a sox10-based fluorescent reporter line for tracing analysis, and images these cells migrating and proliferating in stripes and interstripes^[1]. Why do you think your findings with the pnp4a reporter line are different? Might sox10+ iridophores represent an earlier iridophore progenitor still able to migrate, and pnp4a expression only occur after migration ends?
2. Connected to this, how specific to iridophores is pnp4a? Cell clusters other than iridophores were detected by the RNA-seq analysis, and also express high levels of pteridine and carotenoid-associated genes – could these be xanthophores? Or other cell types entirely?
3. Do you have any idea of the mechanism whereby melanophores might influence iridophore subtype? Do you think xanthophores might also influence iridophore morphology in a similar way?

 

References

1. Singh, A.P., Schach, U., & Nusslein-Volhard, Proliferation, dispersal and patterned aggregation of iridophores in the skin prefigure striped colouration of zebrafish. Nat. Cell. Biol. 16, 607-614
2. Spiewak, J. E. et al. Evolution of Endothelin signaling and diversification of adult pigment pattern in Danio fishes. PLoS Genet 14, e1007538, doi:10.1371/journal.pgen.1007538 (2018).
3. Saunders, L. M. et al. Thyroid hormone regulates distinct paths to maturation in pigment cell lineages. eLife 8, doi:10.7554/eLife.45181 (2019).
4. Frohnhofer, H. G., Krauss, J., Maischein, H. M. & Nusslein-Volhard, C. Iridophores and their interactions with other chromatophores are required for stripe formation in zebrafish. Development 140, 2997-3007, doi:10.1242/dev.096719 (2013).
5. Patterson, L. B. & Parichy, D. M. Interactions with iridophores and the tissue environment required for patterning melanophores and xanthophores during zebrafish adult pigment stripe formation. PLoS Genet 9, e1003561, doi:10.1371/journal.pgen.1003561 (2013).
6. Mahalwar, P., Singh, A. P., Fadeev, A., Nusslein-Volhard, C. & Irion, U. Heterotypic interactions regulate cell shape and density during color pattern formation in zebrafish. Biology open 5, 1680-1690, doi:10.1242/bio.022251 (2016).

 

Tags: melanocytes

doi: https://doi.org/10.1242/prelights.18558

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