Menu

Close

In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning

Dvir Gur, Emily Bain, Kory Johnson, Andy J. Aman, Amalia Pasoili, Jessica D. Flynn, Michael C. Allen, Dimitri D. Deheyn, Jennifer C. Lee, Jennifer Lippincott-Schwartz, David Parichy

Preprint posted on March 25, 2020 https://www.biorxiv.org/content/10.1101/2020.03.25.008664v1

How zebrafish get their stripes – a new model of how iridophores pattern the skin

Selected by Hannah Brunsdon

Categories: developmental biology

 

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

Posted on: 15th April 2020

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

Read preprint (No Ratings Yet)




Author's response

Dave Parichy shared

Q1 – 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?

sox10-Cre labels all neural crest derivatives. So sox10-Cre+ cells were most likely earlier in the lineage, and progenitors to both types. This is interesting because it suggests that single progenitor cells can make (transit-amplifying?) progeny that migrate to the skin and then decide which subtype to differentiate into. Our clonal analyses using pnp4a[2] support this idea. Key in the present manuscript was to use real-time imaging as the cells were developing, and also fate mapping to figure out which cells were already on-hand. There’s just so much proliferation and movement that it’s difficult to work out the event from a single (early) label, even when cells are imaged repeatedly.

Q2 – 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?

pnp4a is most strongly expressed in iridophores but the gene and its reporters are also on at lower levels in melanophores and xanthophores. Single cell RNA-Seq shows this really nicely[3]. This is true as well for some other commonly used reporters. Fortunately, pigment cells have their own autonomous markers (their pigment) and also different morphological hallmarks independent of color, so iridophores expressing pnp4a at high levels, and other pigment cells expressing at lower levels are pretty easy to distinguish.

Q3 – 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?

We are in the midst of testing a couple candidate pathways for melanophores effects on iridophores (and some reciprocal interactions). The Nusslein-Volhard lab has observations that suggest xanthophores may prevent the spread of dense iridophores[4] and, reciprocally, it seems likely that iridophores promote xanthophore differentiation through either or both of a couple mechanisms[5,6]. We tend to think of these and other interactions as a pattern-generating engine; how the engine runs in zebrafish, and how it’s been tweaked in other species with different patterns, are the big questions in the field.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the developmental biology category:

Society for Developmental Biology 79th Annual Meeting

Preprints at SDB 2020

 



List by Irepan Salvador-Martinez, Martin Estermann

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Gautam Dey

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Preprint missing? Don't hesitate to let us know.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

EDBC Alicante 2019

Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.

 



List by Sergio Menchero, Jesus Victorino, Teresa Rayon, Irepan Salvador-Martinez

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Gautam Dey

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve

Pattern formation during development

The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.

 



List by Alexa Sadier

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Gautam Dey

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close