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Understanding how fate specification of distinct cell-types from multipotent progenitors occurs is a fundamental question in embryology. Neural crest stem cells (NCSCs) generate extraordinarily diverse derivatives, including multiple neural, skeletogenic and pigment cell fates. Key transcription factors and extracellular signals specifying NCSC lineages remain to be identified, and we have only a little idea of how and when they function together to control fate. Zebrafish have three neural crest-derived pigment cell types, black melanocytes, light-reflecting iridophores and yellow xanthophores, which offer a powerful model for studying the molecular and cellular mechanisms of fate segregation. Mitfa has been identified as the master regulator of melanocyte fate. Here, we show that an Mitf-related transcription factor, Tfec, functions as master regulator of the iridophore fate. Surprisingly, our phenotypic analysis of tfec mutants demonstrates that Tfec also functions in the initial specification of all three pigment cell-types, although the melanocyte and xanthophore lineages recover later. We show that Mitfa represses tfec expression, revealing a likely mechanism contributing to the decision between melanocyte and iridophore fate. Our data are consistent with the long-standing proposal of a tripotent progenitor restricted to pigment cell fates. Moreover, we investigate activation, maintenance and function of tfec in multipotent NCSCs, demonstrating for the first time its role in the gene regulatory network forming and maintaining early neural crest cells. In summary, we build on our previous work to characterise the gene regulatory network governing iridophore development, establishing Tfec as the master regulator driving iridophore specification from multipotent progenitors, while shedding light on possible cellular mechanisms of progressive fate restriction.

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type.

Over the past decade, CRISPR/Cas-based gene editing has become a powerful tool for generating mutations in a variety of model organisms, from Escherichia coli to zebrafish, rodents and large mammals. CRISPR/Cas-based gene editing effectively generates insertions or deletions (indels), which allow for rapid gene disruption. However, a large proportion of human genetic diseases are caused by single-base-pair substitutions, which result in more subtle alterations to protein function, and which require more complex and precise editing to recreate in model systems. Precise genome editing (PGE) methods, however, typically have efficiencies of less than a tenth of those that generate less-specific indels, and so there has been a great deal of effort to improve PGE efficiency. Such optimisations include optimal guide RNA and mutation-bearing donor DNA template design, modulation of DNA repair pathways that underpin how edits result from Cas-induced cuts, and the development of Cas9 fusion proteins that introduce edits via alternative mechanisms. In this Review, we provide an overview of the recent progress in optimising PGE methods and their potential for generating models of human genetic disease.

Zebrafish are frequently used as a means to investigate development. These studies increasingly require repeated anaesthesia of zebrafish during juvenile (i.e. metamorphic) stages. The effects of anaesthesia during this time remain poorly studied. The aim of this study was to develop a reliable method that can be used for frequently repeated anaesthesia during juvenile stages. Initially, we assessed different concentrations of MS-222, the most commonly used fish anaesthetic, for 30 minute anaesthesia with recovery. We showed that suitable MS-222 doses could be identified for the smallest (7mm) and largest (20mm) fish. However, we found that juvenile fish within a specific metamorphic window (sized between 8–16 mm) were vulnerable to MS-222 and no standard concentration of MS-222 provided reliable anaesthesia under these conditions. Hence we focussed our efforts on identifying a protocol for these stages. We tested six different published anaesthesia protocols P1—P6 where P1, P2 corresponds to 0.01% MS-222, P3, P4: 0.085% 2-phenoxyethanol and P5, P6: 0.00025%/0.0050% Propofol/Lidocaine. In protocols P1, P3, P5 fish were maintained by immersion, whilst in P2, P4 and P6: fish were maintained on an anaesthetic-doused cotton-pad. We assessed reliable anaesthesia using 10 fish for 10 minutes, with full recovery. Our data allowed us to eliminate two of these protocols as unsuitable for short term anaesthesia with recovery of juvenile fish. Extending these studies to explore repeated anaesthesia at 4 day intervals for 20 days under the remaining four protocols, we showed that P1 and P4 were both suitable for repeated anaesthesia, and that P4 was most suitable for imaging. We confirmed that P4 remained suitable when the frequency of anaesthesia was increased to every 2 days. We conclude that this protocol provides a refinement to the current protocol for repeated anaesthesia with recovery of juvenile zebrafish in the vulnerable metamorphic window.

Multipotent neural crest (NC) progenitors generate an astonishing array of derivatives, including neuronal, skeletal components and pigment cells (chromatophores), but the molecular mechanisms allowing balanced selection of each fate remain unknown. In zebrafish, melanocytes, iridophores and xanthophores, the three chromatophore lineages, are thought to share progenitors and so lend themselves to investigating the complex gene regulatory networks (GRNs) underlying fate segregation of NC progenitors. Although the core GRN governing melanocyte specification has been previously established, those guiding iridophore and xanthophore development remain elusive. Here we focus on the iridophore GRN, where mutant phenotypes identify the transcription factors Sox10, Tfec and Mitfa and the receptor tyrosine kinase, Ltk, as key players. Here we present expression data, as well as loss and gain of function results, guiding the derivation of an initial iridophore specification GRN. Moreover, we use an iterative process of mathematical modelling, supplemented with a Monte Carlo screening algorithm suited to the qualitative nature of the experimental data, to allow for rigorous predictive exploration of the GRN dynamics. Predictions were experimentally evaluated and testable hypotheses were derived to construct an improved version of the GRN, which we showed produced outputs consistent with experimentally observed gene expression dynamics. Our study reveals multiple important regulatory features, notably a sox10-dependent positive feedback loop between tfec and ltk driving iridophore specification; the molecular basis of sox10 maintenance throughout iridophore development; and the cooperation between sox10 and tfec in driving expression of pnp4a, a key differentiation gene. We also assess a candidate repressor of mitfa, a melanocyte-specific target of sox10. Surprisingly, our data challenge the reported role of Foxd3, an established mitfa repressor, in iridophore regulation. Our study builds upon our previous systems biology approach, by incorporating physiologically-relevant parameter values and rigorous evaluation of parameter values within a qualitative data framework, to establish for the first time the core GRN guiding specification of the iridophore lineage.

Pattern formation is a key aspect of development. Adult zebrafish exhibit a striking striped pattern generated through the self-organisation of three different chromatophores. Numerous investigations have revealed a multitude of individual cell-cell interactions important for this self-organisation, but it has remained unclear whether these known biological rules were sufficient to explain pattern formation. To test this, we present an individual-based mathematical model incorporating all the important cell-types and known interactions. The model qualitatively and quantitatively reproduces wild type and mutant pigment pattern development. We use it to resolve a number of outstanding biological uncertainties, including the roles of domain growth and the initial iridophore stripe, and to generate hypotheses about the functions of leopard . We conclude that our rule-set is sufficient to recapitulate wild-type and mutant patterns. Our work now leads the way for further in silico exploration of the developmental and evolutionary implications of this pigment patterning system.

Neural crest cells are highly multipotent stem cells, but it remains unclear how their fate restriction to specific fates occurs. The direct fate restriction model hypothesises that migrating cells maintain full multipotency, whilst progressive fate restriction envisages fully multipotent cells transitioning to partially-restricted intermediates before committing to individual fates. Using zebrafish pigment cell development as a model, we show applying NanoString hybridization single cell transcriptional profiling and RNAscope in situ hybridization that neural crest cells retain broad multipotency throughout migration and even in post-migratory cells in vivo, with no evidence for partially-restricted intermediates. We find that leukocyte tyrosine kinase early expression marks a multipotent stage, with signalling driving iridophore differentiation through repression of fate-specific transcription factors for other fates. We reconcile the direct and progressive fate restriction models by proposing that pigment cell development occurs directly, but dynamically, from a highly multipotent state, consistent with our recently-proposed Cyclical Fate Restriction model. Neural crest cells are highly multipotent stem cells, but it remains unclear how their fate restriction to specific fates occurs. Here, the authors show in zebrafish that broad multipotency is retained even after migration, suggesting that fate restriction occurs directly, but dynamically.

Long non-coding RNAs (lncRNAs) play crucial roles in regulating gene expression. Some are essential for organismal development and physiology, and they can contribute to diseases including cancer. Whilst most lncRNAs exhibit little sequence similarity, conservation of lncRNA transcription relative to neighbouring protein-coding genes suggests potential functional significance. Most positionally equivalent lncRNAs are uncharacterized and it remains unclear whether they exert similar roles in distant species. Here, we identified melanoma-associated lncRNAs predicted to be components of the MITF gene regulatory network in human melanoma that have positionally equivalent transcripts in zebrafish. We prioritized the cancer-associated lncRNA Differentiation Antagonizing Non-Protein Coding RNA (DANCR) as an exemplar for functional investigation. DANCR is a multi-exonic, cytoplasmically-enriched lncRNA and small RNA host gene transcribed from syntenic regions in the human and zebrafish genomes. MITF and c-MYC, key melanoma transcription factors, regulate human DANCR expression and melanoma patients with high DANCR display significantly decreased survival. DANCR is a melanoma oncogene that controls cancer-associated gene expression networks to promote human melanoma cell proliferation and migration. Zebrafish dancr is essential for embryonic development. It is dynamically expressed across multiple different cell types in the developing embryo, transcriptionally activated by mitfa during early zebrafish development and it regulates genes involved in cell death. Our work suggests that cancer-critical lncRNAs such as DANCR, expressed from similar regions in vertebrate genomes, may control related genes and processes involved in both embryonic development and tumorigenesis across species.

The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrates is a matter of intense debate. Here, we demonstrate that the non-neuronal ENS cell compartment of teleosts shares molecular and morphological characteristics with mammalian enteric glia but cannot be identified by the expression of canonical glial markers. However, unlike their mammalian counterparts, which are generally quiescent and do not undergo neuronal differentiation during homeostasis, we show that a relatively high proportion of zebrafish enteric glia proliferate under physiological conditions giving rise to progeny that differentiate into enteric neurons. We also provide evidence that, similar to brain neural stem cells, the activation and neuronal differentiation of enteric glia are regulated by Notch signalling. Our experiments reveal remarkable similarities between enteric glia and brain neural stem cells in teleosts and open new possibilities for use of mammalian enteric glia as a potential source of neurons to restore the activity of intestinal neural circuits compromised by injury or disease.

The neural crest (NC) is a multipotent cell population in vertebrate embryos with extraordinary migratory capacity. The NC is crucial for vertebrate development and forms a myriad of cell derivatives throughout the body, including pigment cells, neuronal cells of the peripheral nervous system, cardiomyocytes and skeletogenic cells in craniofacial tissue. NC induction occurs at the end of gastrulation when the multipotent population of NC progenitors emerges in the ectodermal germ layer in the neural plate border region. In the process of NC fate specification, fate-specific markers are expressed in multipotent progenitors, which subsequently adopt a specific fate. Thus, NC cells delaminate from the neural plate border and migrate extensively throughout the embryo until they differentiate into various cell derivatives. Multiple signalling pathways regulate the processes of NC induction and specification. This review explores the ongoing role of the Wnt/β-catenin signalling pathway during NC development, focusing on research undertaken in the Teleost model organism, zebrafish ( Danio rerio ). We discuss the function of the Wnt/β-catenin signalling pathway in inducing the NC within the neural plate border and the specification of melanocytes from the NC. The current understanding of NC development suggests a continual role of Wnt/β-catenin signalling in activating and maintaining the gene regulatory network during NC induction and pigment cell specification. We relate this to emerging models and hypotheses on NC fate restriction. Finally, we highlight the ongoing challenges facing NC research, current gaps in knowledge, and this field’s potential future directions.