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

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Genetic Screen for Postembryonic Development in the Zebrafish (Danio Rerio): Dominant Mutations Affecting Adult Form | INVESTIGATION Genetic Screen for Postembryonic Development in the Zebrafish (Danio rerio): Dominant Mutations Affecting Adult Form Katrin Henke,*,†,1 Jacob M. Daane,*,†,2 M. Brent Hawkins,*,†,‡ Christopher M. Dooley ,§ Elisabeth M. Busch-Nentwich,§,** Derek L. Stemple,§ and Matthew P. Harris*,†,1 *Department of Orthopedic Research, Boston Children’s Hospital, Massachusetts 02115, †Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, ‡Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138, §Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK, and **Department of Medicine, University of Cambridge, CB2 0QQ, UK ABSTRACT 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. KEYWORDS zebrafish; dominant screen; mapping; postembryonic; pigment; skeletogenesis HE use of systematic forward genetic screens has been Many genes that are found to be essential for early develop- Tinstrumental in uncovering genes and pathways involved ment and functional alterations are often not compatible with in a multitude of developmental processes (e.g., Brenner 1974; viability.Such lethality hinders the study of gene function during Nüsslein-Volhard and Wieschaus 1980; Mayer et al. 1991; postembryonic stages. The establishment of tissue-specificor Driever et al. 1996; Haffter et al. 1996a). This phenotype- inducible knock-out lines circumvents this problem, and enables driven approach allows for the unbiased analysis of gene func- analysis in tissues of interest or at specific time-points during tion through generation of random mutations throughout the development. However,these methods do not lend themselves to genome using chemicals or irradiation as mutagens. broad unbiased screening in development, as often only a few loci can be feasibly tested at any one time. Different types of Copyright © 2017 by the Genetics Society of America mutations such as partial loss-of-function or dominant mutations doi: https://doi.org/10.1534/genetics.117.300187 can help in elucidating functions in late development even in Manuscript received June 27, 2017; accepted for publication August 17, 2017; published Early Online August 22, 2017. genes with key roles in embryogenesis. Dominant mutations, in Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10. particular, can be revealing of the full range of molecular and 1534/genetics.117.300187/-/DC1. 1Corresponding authors: Boston Children’s Hospital, Orthopedic Research Laboratories, developmental gene functions, as increased and novel actions of 300 Longwood Ave., Enders 260.2, Mail stop 3096, MA 02115. E-mail: khenke@ a gene can result in unexpected phenotypes. These dominant genetics.med.harvard.edu; and [email protected] 2Present address: Department of Marine and Environmental Sciences, Northeastern mutations can also exhibit dosage-dependent effects, showing University Marine Science Centre, Nahant, MA 01908 graded phenotypic differences between heterozygous and Genetics, Vol. 207, 609–623 October 2017 609 homozygous individuals. Thus, unique mutations apart from affecting the form of the adult zebrafish. To facilitate depth of complete loss-of-function alleles can be informative about molec- screening, we focused on mutants with a dominant effect on ular regulation of gene function in postembryonic development. morphology. Importantly, unlike most alleles identified or cre- The zebrafish is a well-established genetic model. Screens ated using genome editing techniques, our focus on dominant have focused on the identification of genes important for early mutations was centered on the abilitytoprovideinsightinto the developmental processes, with mutants showing recessive molecular action of a gene other than simple loss-of-function inheritance of the phenotype (e.g., Driever et al. 1996; alleles through identification of potential gain-of-function and Haffter et al. 1996a). Screens for recessive mutations require neomorphic alleles. Here, we show the feasibility of large-scale breeding the induced mutations to homozygosity and there- dominant screens in zebrafish for postembryonic development, fore multiple generations need to be raised before a pheno- including methods to systematically identify linkage and caus- type is visible in the F3 generation. Thus, recessive screens ative mutations underlying dominant mutant phenotypes. Re- require the ability to raise and screen a large number of fish in sults from this screen define important disease models in the order to screen the function of genes affecting a specific de- zebrafish. Furthermore, by clustering analysis of similar phe- velopmental process in sufficient depth. notypes, we show enrichment for mutations affecting extracel- Themajorityoftheidentified recessive mutants display early lular matrix formation as a regulator of late development. phenotypes and are embryonic or larval lethal. Only 3% of Importantly, this screen sets the stage for the use of zebrafish mutants identified in these screens for early larval phenotypes as an experimental tool to investigate genetic regulation led to viable adults with observable phenotypes (Haffter et al. through modifier analysis and efficient identification of gene 1996a). Thus, much of the genetic regulation of late develop- networks regulating late development. ment remains undescribed. Few screens have looked for genes affecting late development and most have been restricted in Materials and Methods depth and phenotypic breadth (Haffter et al. 1996b; Bauer fi and Goetz 2001; Fisher et al. 2003; Andreeva et al. 2011; Husbandry and management of identi ed mutant lines fi Saito et al. 2011). However, larger screens looking speci cally Zebrafish (Danio rerio) were raised and maintained under stan- for genes necessary for normal patterning and growth of dard conditions (Nüsslein-Volhard and Dahm 2002) in compli- fi the adult sh, demonstrated that a large number of mutants ance with internal regulatory review at Boston Children’s fi could be identi ed, supporting that late developmental processes Hospital. Mutant lines were named following the rules set out can be investigated by classic genetic approaches (ZF models; by ZFIN, where “mh” is the designation of the founding lab www.zf-health.org/zf-models). In comparison, the largest of (Harris lab) and “d” indicates dominant inheritance of the allele. these screens from the ZF models consortium scored 1000 genomes for adult traits, 1/6 of the total predicted number Mutagenesis and screen design of genomes analyzed for larval phenotypes in this screen, as well To identify dominant mutations that affect the adult form of as the number of combined genomes screened in the early the zebrafish, mutations were induced by treatment of zygotic screens (Driever et al. 1996; Haffter et al. 1996a). In- 30 wild-type Tübingen males with N-ethyl-N-nitrosourea triguingly, many of the mutations identified from these adult (ENU) treatment following an optimized protocol (Rohner screens affect genes whose orthologs are associated with disease et al. 2011). The surviving 14 mutagenized males were in humans (fgfr1a, col1a1a, bmp1a,andedar) (Fisher et al. 2003; mated twice a week with wild-type females, and over Harris et al. 2008; Rohner et al. 2009; Asharani et al. 2012). 14,000 progeny were raised. At 10–12 weeks postfertiliza- While isolating mutant lines through genetic screens has been tion (wpf), F1 fish were anesthetized in 0.02% MS-222 and very successful, identification of the causative mutations un- screened under a dissecting scope for morphological changes. derlying mutant phenotypes was previously difficult, limiting the Potential mutants were isolated and crossed to wild-type fish. broad analysis of large classes of mutants and preventing detail- Similarly, F2 progeny were screened at 10–12 wpf. Crosses ing of their cognizant genetic pathways. The advent of next-
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