Christopher M. Dooley

A systematic genome-wide analysis of zebrafish protein-coding gene function

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

Slc45a2 and V‐ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease

We present here the positional cloning of the Danio rerio albino mutant and show that the affected gene encodes Slc45a2. The human orthologous gene has previously been shown to be involved in human skin color variation, and mutations therein have been implicated in the disease OCA4. Through ultrastructural analysis of the melanosomes in albino alleles as well as the tyrosinase‐deficient mutant sandy, we add new insights into the role of Slc45a2 in the production of melanin. To gain further understanding of the role of Slc45a2 and its possible interactions with other proteins involved in melanization, we further analyzed the role of the V‐ATPase as a melanosomal acidifier. We show that it is possible to rescue the melanization potential of the albino melanosomes through genetic and chemical inhibition of V‐ATPase, thereby increasing internal melanosome pH.

On the embryonic origin of adult melanophores: the role of ErbB and Kit signalling in establishing melanophore stem cells in zebrafish

Pigment cells in vertebrates are derived from the neural crest (NC), a pluripotent and migratory embryonic cell population. In fishes, larval melanophores develop during embryogenesis directly from NC cells migrating along dorsolateral and ventromedial paths. The embryonic origin of the melanophores that emerge during juvenile development in the skin to contribute to the striking colour patterns of adult fishes remains elusive. We have identified a small set of melanophore progenitor cells (MPs) in the zebrafish (Danio rerio, Cyprinidae) that is established within the first 2 days of embryonic development in close association with the segmentally reiterated dorsal root ganglia (DRGs). Lineage analysis and 4D in vivo imaging indicate that progeny of these embryonic MPs spread segmentally, giving rise to the melanophores that create the adult melanophore stripes. Upon depletion of larval melanophores by morpholino knockdown of Mitfa, the embryonic MPs are prematurely activated; their progeny migrate along the spinal nerves restoring the larval pattern and giving rise to postembryonic MPs associated with the spinal nerves. Mutational or chemical inhibition of ErbB receptors blocks all early NC migration along the ventromedial path, causing a loss of DRGs and embryonic MPs. We show that the sparse like (slk) mutant lacks larval and metamorphic melanophores and identify kit ligand a (kitlga) as the underlying gene. Our data suggest that kitlga is required for the establishment or survival of embryonic MPs. We propose a model in which DRGs provide a niche for the stem cells of adult melanophores.

Large-scale mapping of mutations affecting zebrafish development

BackgroundLarge-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.ResultsWe have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.ConclusionBy obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.

Multi-allelic phenotyping--a systematic approach for the simultaneous analysis of multiple induced mutations.

The zebrafish mutation project (ZMP) aims to generate a loss of function allele for every protein-coding gene, but importantly to also characterise the phenotypes of these alleles during the first five days of development. Such a large-scale screen requires a systematic approach both to identifying phenotypes, and also to linking those phenotypes to specific mutations. This phenotyping pipeline simultaneously assesses the consequences of multiple alleles in a two-step process. First, mutations that do not produce a visible phenotype during the first five days of development are identified, while a second round of phenotyping focuses on detailed analysis of those alleles that are suspected to cause a phenotype. Allele-specific PCR single nucleotide polymorphism (SNP) assays are used to genotype F2 parents and individual F3 fry for mutations known to be present in the F1 founder. With this method specific phenotypes can be linked to induced mutations. In addition a method is described for cryopreserving sperm samples of mutagenised males and their subsequent use for in vitro fertilisation to generate F2 families for phenotyping. Ultimately this approach will lead to the functional annotation of the zebrafish genome, which will deepen our understanding of gene function in development and disease.

KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1.

Abstract Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing.

A high-resolution mRNA expression time course of embryonic development in zebrafish

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3′ end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3′ ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.

High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

BackgroundWe present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements.ResultsMultiplex sequencing of transcript 3′ ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts.ConclusionsThis method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.

Genetic Screen for Postembryonic Development in the Zebrafish (Danio rerio): Dominant Mutations Affecting Adult Form

Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, Danio rerio, is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.

Leucophore identity is more gold than silver.

The diversity of body colour patterns seen throughout nature arises from neural crest-derived chromatophores. Mammals and birds achieve a stunning variety of colour with a single chromatophore of the body, the melanocyte. Our cold-blooded relatives, fish, amphibians and reptiles typically have a larger palette to choose from, presenting most commonly with melanocytes, xanthophores and iridophores. Medaka are one of the minority of fish species which also form a forth white reflective cell type called the leucophore. The reflective nature of leucophore’s purinerelated, uric acid intracellular pigment elements had previously lead to the conclusion that leucophores were more closely related to silvery iridophores. Recently, however, both Nagao et al., and Kimura et al., make compelling cases for leucophores sharing a neural crest lineage with xanthophores instead. Exactly, how the neural crest gene regulatory network achieves specific cell fates has been intensely investigated over the years. The molecular mechanisms required in fate specification of melanocytes are relatively well understood and a shared lineage of melanophores and iridophores seems likely in zebrafish (Budi et al., 2011; Singh et al., 2014). Kimura et al., have identified the underlying causative genes for three leucophore mutants: leucophore-free/slc2a 15b, lf-2/pax7a and white leucophore/slc2a11b (Kimura et al., 2014). All three mutants affect both leucophores and xanthophores initially highlighting the developmental relationship of the two chromatophores. Closer inspection reveals a more structured hierarchy where pax7a expression in neural crest cells precedes the appearance of all chromatophores and is required for the development of both leucophores and xanthophores in the trunk. Leucophorefree/slc2a15b mutants were shown to successfully establish leucophore and xanthophores precursor cells, but these cells failed to differentiate and pigment properly. White leucophore/slc2a11b mutants were also able to form both leucophore and xanthophore precursor cells, but these cells were defective in their ability to produce orange or yellow pigmentation. A phylogenetic tree based on the amino acid sequence of SLC2A class II transporters using ascidian and vertebrate orthologs also revealed a coupled presence of slc2a15b and slc2a11b in other animals presenting xanthophores. With a close developmental relationship between leucophores and xanthophores becoming clear, the ensuing crucial question remains what molecular switches are required to specify leucophores and xanthophores from their common precursor? As Nagao et al. (2014) nicely demonstrate that switch is governed by sox5. The many leucophores-3/sox5 mutants lack all visible xanthophores in both embryonic and larval stage medaka but have an expanded population of leucophores. Many leucophores-3/sox5 heterozygous fish also develop an increased population of leucophores and have a noticeable reduction in xanthophores. The semidominant nature of the phenotype points towards a sox5 dose-dependent relationship between the two chromatophores. Using reciprocating cell transplantation experiments between many leucophores-3/sox5 and wild-type embryos, it was revealed that the sox5 switch between leucophores and xanthophores works in a cell autonomous fashion. So, if sox5 is the switch, when is it used? Both leucophores and xanthophores depend on pax7a for the specification of their common precursors. Xanthophores then require the expression of sox5 whereas leucophores do not as lack of sox5 in many leucophores-3 leads to a complete shift to the leucophore lineage at the expense of xanthophores. With four chromatophore linages to be determined in medaka, what role does sox5 play in the lineage of xanthophores in fish species with only three chromatophore types? Does sox5 have other roles in the neural crest cells of animals completely lacking xanthophores? Armed with these new questions and a more detailed understanding of specific neural crest fate switches, it is only a matter of time.