Genetics: Early Online, published on August 23, 2017 as 10.1534/genetics.117.300187 Genetic screen for post-embryonic 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‡, Matthew P. Harris*,1 *Department of Orthopedic Surgery Research, Boston Children’s Hospital; Department of Genetics, Harvard Medical School, Boston, MA 02115 !Department of Organismic and Evolutionary Biology, Harvard University, Cambridge MA, 02138 ‡Wellcome Trust Sanger Institute, Hinxton, UK CB101SA §Department of Medicine, University of Cambridge, Cambridge, UK 1 to whom correspondence should be sent, [email protected], [email protected] 2Current address: Department of Marine and Environmental Sciences, Northeastern University Marine Science Centre, Nahant, MA 01908 1 Copyright 2017. Abstract Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, 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 post-embryonic 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 post-embryonic 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 post-embryonic development and in disease. 2 Introduction The use of systematic forward genetic screens has been instrumental in uncovering genes and pathways involved in a multitude of developmental processes (e.g. Brenner 1974; Nüsslein-Volhard and Wieschaus 1980; Mayer et al. 1991; Haffter, Granato, et al. 1996; Driever et al. 1996). This phenotype driven approach allows for the unbiased analysis of gene function through generation of random mutations throughout the genome using chemicals or irradiation as mutagens. Many genes that are found to be essential for early development and functional alterations are often not compatible with viability. Such lethality hinders the study of gene function during postembryonic stages. The establishment of tissue specific or inducible knock-out lines circumvents this problem and enables analysis in tissues of interest or at specific time-points during development. However, these methods do not lend themselves to broad unbiased screening in development, as often only a few loci can be feasibly tested at any one time. Different types of mutations such as partial loss-of-function or dominant mutations can help in elucidating functions in late development even in genes with key roles in embryogenesis. Dominant mutations, in particular, can be revealing of the full range of molecular and developmental gene functions, as increased and novel actions of a gene can result in unexpected phenotypes. These dominant mutations can also exhibit dosage dependent effects, showing graded phenotypic differences between heterozygous and homozygous individuals. Thus, unique mutations apart from complete loss-of-function alleles can be informative about molecular regulation of gene function in post-embryonic development. The zebrafish is a well-established genetic model. Screens have focused on the identification of genes important for early developmental processes, with mutants showing recessive inheritance of the phenotype (e.g. Haffter, Granato, et al. 1996; Driever et al. 1996). Screens for recessive mutations require breeding the induced mutations to homozygosity and therefore multiple generations need to be raised before a phenotype is visible in the F3 generation. Thus, recessive screens require the ability to raise and screen a large number of fish in order to screen the function of genes affecting a specific developmental process in sufficient depth. 3 The majority of the identified recessive mutants display early phenotypes and are embryonic or larval lethal. Only about 3% of mutants identified in these screens for early larval phenotypes led to viable adults with observable phenotypes (Haffter, Granato, et al. 1996). Thus, much of the genetic regulation of late development remains undescribed. Few screens have looked for genes affecting late development and most have been restricted in depth and phenotypic breadth (Haffter, Odenthal, et al. 1996; Bauer and Goetz 2001; Fisher, Jagadeeswaran, and Halpern 2003; Andreeva et al. 2011; Saito et al. 2011). However, larger screens looking specifically for genes necessary for normal patterning and growth of the adult fish, demonstrated that a large number of mutants could be identified, supporting that late developmental processes can be investigated by classic genetic approaches (ZF models; www.zf-health.org/zf-models). In comparison, the largest of these screens from the ZF models consortium scored about 1000 genomes for adult traits, about 1/6 of the total predicted number of genomes analyzed for larval phenotypes in this screen as well as the number of combined genomes screened in the early zygotic screens (Haffter, Granato, et al. 1996; Driever et al. 1996). Intriguingly, many of the mutations identified from these adult screens affect genes whose orthologues are associated with disease in humans (fgfr1a, col1a1a, bmp1a, edar) (Rohner et al. 2009; Fisher, Jagadeeswaran, and Halpern 2003; Asharani et al. 2012, 1; Harris et al. 2008). While isolating mutant lines through genetic screens has been very successful, identification of the causative mutations underlying mutant phenotypes was previously difficult, limiting the broad analysis of large classes of mutants and preventing detailing of their cognizant genetic pathways. The advent of next-generation sequencing techniques in combination with targeted capture and multiplexing of samples has allowed for cost- effective and efficient identification of mutations (Bowen et al. 2012; Leshchiner et al. 2012; Voz et al. 2012; Obholzer et al. 2012; Henke, Bowen, and Harris 2013; Kettleborough et al. 2013; Ryan et al. 2013). This has opened the potential for analysis of genetic networks regulating specific developmental processes, as linkage and potential causative mutations can be quickly defined in whole sets of mutants. We sought to take advantage of these new sequencing technologies to explore the utility of zebrafish genetic screens to investigate the genes controlling post-embryonic 4 development. Towards this end, we initiated a screen for mutations affecting the form of the adult zebrafish. To facilitate depth of screening, we focused on mutants with a dominant effect on morphology. Importantly, unlike most alleles identified or created using genome editing techniques, our focus on dominant mutations was centered on the ability to provide insight into the molecular action of a gene other than simple loss-of-function alleles through identification of potential gain-of-function and neomorphic alleles. Here we show the feasibility of large-scale dominant screens in zebrafish for post-embryonic development, including methods to systematically identify linkage and causative mutations underlying dominant mutant phenotypes. Results from this screen define important disease models in the zebrafish. Furthermore, by clustering analysis of similar phenotypes we show enrichment for mutations affecting extracellular matrix formation as a regulator of late development. Importantly, this screen sets the stage for the use of zebrafish as an experimental tool to investigate genetic regulation through modifier analysis and efficient identification of gene networks regulating late development. Methods Husbandry and management of identified mutant lines Zebrafish (Danio rerio) were raised and maintained under standard conditions (Nüsslein- Volhard and Dahm 2002) in compliance with internal regulatory review at Boston Children’s Hospital. Mutant lines were named following the rules set out by ZFIN, where ‘mh’ is the designation of the founding lab (Harris lab) and ‘d’ indicates dominant inheritance of the allele. Mutagenesis and screen design To identify dominant mutations that affect the adult form of the zebrafish, mutations were induced by treatment of 30 wildtype Tübingen males with N-ethyl-N-nitrosourea (ENU) treatment following an optimized protocol (Rohner et al. 2011). The surviving 14 mutagenized males were mated twice a week