Lysophosphatidic acid signaling via LPA6 : A negative modulator of developmental oligodendrocyte maturation.

The developmental process of central nervous system (CNS) myelin sheath formation is characterized by well-coordinated cellular activities ultimately ensuring rapid and synchronized neural communication. During this process, myelinating CNS cells, namely oligodendrocytes (OLGs), undergo distinct steps of differentiation, whereby the progression of earlier maturation stages of OLGs represents a critical step toward the timely establishment of myelinated axonal circuits. Given the complexity of functional integration, it is not surprising that OLG maturation is controlled by a yet fully to be defined set of both negative and positive modulators. In this context, we provide here first evidence for a role of lysophosphatidic acid (LPA) signaling via the G protein-coupled receptor LPA6 as a negative modulatory regulator of myelination-associated gene expression in OLGs. More specifically, the cell surface accessibility of LPA6 was found to be restricted to the earlier maturation stages of differentiating OLGs, and OLG maturation was found to occur precociously in Lpar6 knockout mice. To further substantiate these findings, a novel small molecule ligand with selectivity for preferentially LPA6 and LPA6 agonist characteristics was functionally characterized in vitro in primary cultures of rat OLGs and in vivo in the developing zebrafish. Utilizing this approach, a negative modulatory role of LPA6 signaling in OLG maturation could be corroborated. During development, such a functional role of LPA6 signaling likely serves to ensure timely coordination of circuit formation and myelination. Under pathological conditions as seen in the major human demyelinating disease multiple sclerosis (MS), however, persistent LPA6 expression and signaling in OLGs can be seen as an inhibitor of myelin repair. Thus, it is of interest that LPA6 protein levels appear elevated in MS brain samples, thereby suggesting that LPA6 signaling may represent a potential new druggable pathway suitable to promote myelin repair in MS.

The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor.

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.

Mitf-family transcription factor function is required within cranial neural crest cells to promote choroid fissure closure.

A crucial step in eye development is the closure of the choroid fissure (CF), a transient structure in the ventral optic cup through which vasculature enters the eye and ganglion cell axons exit. Although many factors have been identified that function during CF closure, the molecular and cellular mechanisms mediating this process remain poorly understood. Failure of CF closure results in colobomas. Recently, MITF was shown to be mutated in a subset of individuals with colobomas, but how MITF functions during CF closure is unknown. To address this issue, zebrafish with mutations in mitfa and tfec, two members of the Mitf family of transcription factors, were analyzed and their functions during CF closure determined. mitfa;tfec mutants possess severe colobomas and our data demonstrate that Mitf activity is required within cranial neural crest cells (cNCCs) during CF closure. In the absence of Mitf function, cNCC migration and localization in the optic cup are perturbed. These data shed light on the cellular mechanisms underlying colobomas in individuals with MITF mutations and identify a novel role for Mitf function in cNCCs during CF closure.

Zebrafish MITF-Low Melanoma Subtype Models Reveal Transcriptional Subclusters and MITF-Independent Residual Disease.

The melanocyte-inducing transcription factor (MITF)-low melanoma transcriptional signature is predictive of poor outcomes for patients, but little is known about its biological significance, and animal models are lacking. Here, we used zebrafish genetic models with low activity of Mitfa (MITF-low) and established that the MITF-low state is causal of melanoma progression and a predictor of melanoma biological subtype. MITF-low zebrafish melanomas resembled human MITF-low melanomas and were enriched for stem and invasive (mesenchymal) gene signatures. MITF-low activity coupled with a p53 mutation was sufficient to promote superficial growth melanomas, whereas BRAFV600E accelerated MITF-low melanoma onset and further promoted the development of MITF-high nodular growth melanomas. Genetic inhibition of MITF activity led to rapid regression; recurrence occurred following reactivation of MITF. At the regression site, there was minimal residual disease that was resistant to loss of MITF activity (termed MITF-independent cells) with very low-to-no MITF activity or protein. Transcriptomic analysis of MITF-independent residual disease showed enrichment of mesenchymal and neural crest stem cell signatures similar to human therapy-resistant melanomas. Single-cell RNA sequencing revealed MITF-independent residual disease was heterogeneous depending on melanoma subtype. Further, there was a shared subpopulation of residual disease cells that was enriched for a neural crest G0-like state that preexisted in the primary tumor and remained present in recurring melanomas. These findings suggest that invasive and stem-like programs coupled with cellular heterogeneity contribute to poor outcomes for MITF-low melanoma patients and that MITF-independent subpopulations are an important therapeutic target to achieve long-term survival outcomes. SIGNIFICANCE: This study provides a useful model for MITF-low melanomas and MITF-independent cell populations that can be used to study the mechanisms that drive these tumors as well as identify potential therapeutic options.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/22/5769/F1.large.jpg.

Larval but not adult xanthophore pigmentation in zebrafish requires GTP cyclohydrolase 2 (gch2) function.

Although xanthophores are found widely among poikilothermic species, the developmental and biochemical pathways underlying differentiation of these pteridine- and carotenoid-containing cells remain murky. I have identified a recessive zebrafish mutant, camembert (cmm), which displays defective xanthophore pigmentation during embryonic and larval stages with cells appearing grayish rather than yellow, but as an adult appears to have normally pigmented xanthophores and wild-type stripe pattern. Examination of molecular markers reveals that xanthophores are present in typical numbers and position in cmm embryos; however, the localization of transcripts for the gene GTP cyclohydrolase 2 (gch2), encoding a critical protein in the pteridine biosynthetic pathway, is strikingly altered. RT-PCR analysis indicates that gch2 transcripts in mutant embryos skip an exon or retain the intron upstream and that no correctly spliced mRNA is made. Sequencing of genomic DNA reveals that the skipped exon is intact, but the retained intron contains a deletion of approximately 180 base pairs, just upstream of the splice acceptor. Microinjection of a gch2 BAC clone rescues yellow pigmentation in camembert larvae, confirming that the pigmentation defect is due to mutation of gch2.

A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest.

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.

Melanoma Regression and Recurrence in Zebrafish.

Melanoma is the most lethal form of skin cancer with high mortality rates. Most melanoma cases have activating mutations in BRAF (V600E) and the selective inhibitors of BRAF(V600E) have been successfully used in patients. However, after initial tumor regression, the majority of patients develop drug resistance resulting in tumor regrowth. It is therefore important to understand the mechanisms underlying these processes. We have recently described the role of the master melanocyte transcription factor MITF in tumor growth, regression, and recurrence. Here, we describe protocols to study regression and recurrence in vivo, as well as for histology and immunohistochemistry, using a temperature-sensitive zebrafish model of human melanoma.

The Autotaxin-Lysophosphatidic Acid Axis Modulates Histone Acetylation and Gene Expression during Oligodendrocyte Differentiation.

During development, oligodendrocytes (OLGs), the myelinating cells of the CNS, undergo a stepwise progression during which OLG progenitors, specified from neural stem/progenitor cells, differentiate into fully mature myelinating OLGs. This progression along the OLG lineage is characterized by well synchronized changes in morphology and gene expression patterns. The latter have been found to be particularly critical during the early stages of the lineage, and they have been well described to be regulated by epigenetic mechanisms, especially by the activity of the histone deacetylases HDAC1 and HDAC2. The data presented here identify the extracellular factor autotaxin (ATX) as a novel upstream signal modulating HDAC1/2 activity and gene expression in cells of the OLG lineage. Using the zebrafish as an in vivo model system as well as rodent primary OLG cultures, this functional property of ATX was found to be mediated by its lysophospholipase D (lysoPLD) activity, which has been well characterized to generate the lipid signaling molecule lysophosphatidic acid (LPA). More specifically, the lysoPLD activity of ATX was found to modulate HDAC1/2 regulated gene expression during a time window coinciding with the transition from OLG progenitor to early differentiating OLG. In contrast, HDAC1/2 regulated gene expression during the transition from neural stem/progenitor to OLG progenitor appeared unaffected by ATX and its lysoPLD activity. Thus, together, our data suggest that an ATX-LPA-HDAC1/2 axis regulates OLG differentiation specifically during the transition from OLG progenitor to early differentiating OLG and via a molecular mechanism that is evolutionarily conserved from at least zebrafish to rodent. SIGNIFICANCE STATEMENT: The formation of the axon insulating and supporting myelin sheath by differentiating oligodendrocytes (OLGs) in the CNS is considered an essential step during vertebrate development. In addition, loss and/or dysfunction of the myelin sheath has been associated with a variety of neurologic diseases in which repair is limited, despite the presence of progenitor cells with the potential to differentiate into myelinating OLGs. This study characterizes the autotaxin-lysophosphatidic acid signaling axis as a modulator of OLG differentiation in vivo in the developing zebrafish and in vitro in rodent OLGs in culture. These findings provide novel insight into the regulation of developmental myelination, and they are likely to lead to advancing studies related to the stimulation of myelin repair under pathologic conditions.

A conditional zebrafish MITF mutation reveals MITF levels are critical for melanoma promotion vs. regression in vivo.

The microphthalmia-associated transcription factor (MITF) is the "master melanocyte transcription factor" with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAF(V600E) to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAF(V600E)mitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

Otx but not Mitf transcription factors are required for zebrafish retinal pigment epithelium development.

Otx and Mitf transcription factors have been implicated in the development of the retinal pigmented epithelium (RPE), but the relationship between these factors and their specific roles in the development of the RPE have not been fully defined. The role of the three Otx transcription factors (Otx1a, Otx1b, and Otx2) and two Mitf transcription factors (Mitfa and Mitfb) in the development of the zebrafish RPE was explored in these experiments. The loss of Otx activity through morpholino knockdown produced variable eye defects, ranging from delayed RPE pigmentation to severe coloboma, depending on the combination of Otx factors that were targeted. Expression analysis through in situ hybridization demonstrates that otx transcription factors are necessary for the proper expression of mitfa and mitfb while Mitf transcription factors are not required for the expression of otx genes. Surprisingly, the loss of Mitf activity in mitfa, mitfb, or double mitf mutant zebrafish had no effect on RPE pigmentation or development. Moreover, histological analysis revealed that retinal lamination is unaffected in mitf mutants, as well as in otx morphants, even in regions lacking RPE. Otx and Mitf combined loss of function experiments suggest that mitfa and mitfb may still influence zebrafish RPE development. This is further supported by the ability of mitfa to induce pigmentation in the zebrafish retina when misexpressed. These findings suggest that one or more Otx targets in addition to mitfa and mitfb, possibly another mitf family member, are necessary for development of the RPE in zebrafish.

Autotaxin/ENPP2 regulates oligodendrocyte differentiation in vivo in the developing zebrafish hindbrain.

During development, progenitors that are committed to differentiate into oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated within discrete regions of the neuroepithelium. More specifically, within the developing spinal cord and hindbrain ventrally located progenitor cells that are characterized by the expression of the transcription factor olig2 give temporally rise to first motor neurons and then oligodendrocyte progenitors. The regulation of this temporal neuron-glial switch has been found complex and little is known about the extrinsic factors regulating it. Our studies described here identified a zebrafish ortholog to mammalian atx, which displays evolutionarily conserved expression pattern characteristics. Most interestingly, atx was found to be expressed by cells of the cephalic floor plate during a time period when ventrally-derived oligodendrocyte progenitors arise in the developing hindbrain of the zebrafish. Knock-down of atx expression resulted in a delay and/or inhibition of the timely appearance of oligodendrocyte progenitors and subsequent developmental stages of the oligodendrocyte lineage. This effect of atx knock-down was not accompanied by changes in the number of olig2-positive progenitor cells, the overall morphology of the axonal network or the number of somatic abducens motor neurons. Thus, our studies identified Atx as an extrinsic factor that is likely secreted by cells from the floor plate and that is involved in regulating specifically the progression of olig2-positive progenitor cells into lineage committed oligodendrocyte progenitors.

Use of phage φC31 integrase as a tool for zebrafish genome manipulation.

On the strengths of forward genetics and embryology, the zebrafish Danio rerio has become an ideal system for the study of early vertebrate development. However, additional tools will be needed to perform more sophisticated analyses and to successfully carry this model into new areas of study such as adult physiology, cancer, and aging. As improved tools make transgenesis more and more efficient, the stage has been set for precise modification of the zebrafish genome such as are done in other model organisms. Genome engineering strategies employing site-specific recombinase (SSR) systems such as Cre/lox and Flp/FRT have become invaluable to the study of gene function in the mouse and Drosophila and are now being exploited in zebrafish as well. My laboratory has begun to use another such SSR, the integrase encoded by the Streptomyces bacteriophage PhiC31, for manipulation of the zebrafish genome. The PhiC31 integrase promotes recombination between an attachment site in the phage (attP) and another on the bacterial chromosome (attB). Here I describe strategies using the PhiC31 integrase to mediate recombination of transgenes containing attP and attB sites in cis to excise elements with spatial and temporal specificity. The feasibility of the intramolecular recombination approach having been established, I discuss prospects for employing PhiC31 integrase for intermolecular recombination, i.e., transgene integration at defined sites in the genome.

Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec.

The MiT family comprises four genes in mammals: Mitf, Tfe3, Tfeb, and Tfec, which encode transcription factors of the basic-helix-loop-helix/leucine zipper class. Mitf is well-known for its essential role in the development of melanocytes, however the functions of the other members of this family, and of interactions between them, are less well understood. We have now characterized the complete set of MiT genes from zebrafish, which totals six instead of four. The zebrafish genome contain two mitf (mitfa and mitfb), two tfe3 (tfe3a and tfe3b), and single tfeb and tfec genes; this distribution is shared with other teleosts. We present here the sequence and embryonic expression patterns for the zebrafish tfe3b, tfeb, and tfec genes, and identify a new isoform of tfe3a. These findings will assist in elucidating the roles of the MiT gene family over the course of vertebrate evolution.

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf.

Coordination of cell proliferation and differentiation is crucial for tissue formation, repair and regeneration. Some tissues, such as skin and blood, depend on differentiation of a pluripotent stem cell population, whereas others depend on the division of differentiated cells. In development and in the hair follicle, pigmented melanocytes are derived from undifferentiated precursor cells or stem cells. However, differentiated melanocytes may also have proliferative capacity in animals, and the potential for differentiated melanocyte cell division in development and regeneration remains largely unexplored. Here, we use time-lapse imaging of the developing zebrafish to show that while most melanocytes arise from undifferentiated precursor cells, an unexpected subpopulation of differentiated melanocytes arises by cell division. Depletion of the overall melanocyte population triggers a regeneration phase in which differentiated melanocyte division is significantly enhanced, particularly in young differentiated melanocytes. Additionally, we find reduced levels of Mitf activity using an mitfa temperature-sensitive line results in a dramatic increase in differentiated melanocyte cell division. This supports models that in addition to promoting differentiation, Mitf also promotes withdrawal from the cell cycle. We suggest differentiated cell division is relevant to melanoma progression because the human melanoma mutation MITF(4T)(Δ)(2B) promotes increased and serial differentiated melanocyte division in zebrafish. These results reveal a novel pathway of differentiated melanocyte division in vivo, and that Mitf activity is essential for maintaining cell cycle arrest in differentiated melanocytes.

mitfa is required at multiple stages of melanocyte differentiation but not to establish the melanocyte stem cell.

The mitfa gene encodes a zebrafish ortholog of the microphthalmia-associated transcription factor (Mitf) which, like its counterparts in other species, is absolutely required for development of neural crest melanocytes. In order to evaluate mitfa's role in different stages of melanocyte development, we have identified hypomorphic alleles of mitfa, including two alleles that are temperature-sensitive for melanocyte development. Molecular analysis revealed that the mitf(fh53)ts results from a single base pair change producing an asparagine to tyrosine amino acid substitution in the DNA-binding domain, and the mitfa(vc7)ts allele is a mutation in a splice donor site that reduces the level of correctly-spliced transcripts. Splicing in the mitfa(vc7) allele does not itself appear to be temperature-dependent. A third, hypomorphic allele, mitfa(z25) results in an isoleucine to phenylalanine substitution in the first helix domain of the protein. Temperature upshift experiments with mitfa(fh53)ts show that mitfa is required at several stages of melanocyte differentiation, including for expression of the early melanoblast marker dct, again for progression from dct expression to differentiation, and again for maintenance of dendritic form following differentiation. mitfa(fh53)ts mutants recover melanocytes within 2-3days when downshifted at all stages of larval development. However, when melanocyte stem cells (MSCs) are ablated by early treatment with the erbB3 inhibitor AG1478, melanocyte recovery is lost by 48 h. This result indicates first that the MSC is established at the restrictive temperature, and that melanoblasts die or lose the ability to recover after being held at the restrictive temperature for approximately one day.

Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.

Pigment cells of the zebrafish, Danio rerio, offer an exceptionally tractable system for studying the genetic and cellular bases of cell fate decisions. In the zebrafish, neural crest cells generate three types of pigment cells during embryogenesis: yellow xanthophores, iridescent iridophores and black melanophores. In this study, we present evidence for a model whereby melanophores and iridophores descend from a common precursor whose fate is regulated by an interplay between the transcription factors Mitf and Foxd3. Loss of mitfa, a key regulator of melanophore development, resulted in supernumerary ectopic iridophores while loss of foxd3, a mitfa repressor, resulted in fewer iridophores. Double mutants showed a restoration of iridophores, suggesting that one of Foxd3's roles is to suppress mitfa to promote iridophore development. Foxd3 co-localized with pnp4a, a novel marker of early iridophore development, and was necessary for its expression. A considerable overlap was found between iridoblast and melanoblast markers but not xanthoblast markers, which resolved as cells began to differentiate. Cell lineage analyses using the photoconvertible marker, EosFP, revealed that both melanophores and iridophores develop from a mitfa+ precursor. Taken together, our data reveal a Foxd3/mitfa transcriptional switch that governs whether a bi-potent pigment precursor will attain either an iridophore or a melanophore fate.

Transgene excision in zebrafish using the phiC31 integrase.

Genome engineering strategies employing site-specific recombinases (SSRs) have become invaluable to the study of gene function in model organisms. One such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, promotes recombination between heterotypic attP and attB sites. In the present study I have examined the feasibility of the use of phiC31 integrase for intramolecular recombination strategies in zebrafish embryos. I report here that (1) phiC31 integrase is functional in zebrafish cells, (2) phiC31 integrase can excise a transgene cassette flanked by an attB and an attP site, analogous to a common use of the Cre/lox SSR system, (3) phiC31 integrase functions in the zebrafish germline, and (4) a phiC31 integrase-estrogen receptor hormone-binding domain variant fusion protein catalyzes attB-attP recombination in zebrafish embryos in a 4-hydroxytamoxifen-dependent manner, albeit less efficiently than phiC31 alone. These features should make this a useful approach for genome manipulations in the zebrafish.