Narratives of recovery from mental health distress have played a central role in the establishment of the recovery paradigm within mental health policy and practice. As use of recovery narratives increases within services, it is critical to understand how they have been characterised, and what may be missing from their characterisation thus far. The aim of this review was to synthesise published typologies in order to develop a conceptual framework characterising mental health recovery narratives.A systematic review was conducted of published literature on the characteristics of mental health recovery narratives. Narrative synthesis involved identifying characteristics and organising them into dimensions and types; and subgroup analysis based on study quality, narrator involvement in analysis, diagnosis of psychosis and experience of trauma. The synthesis was informed by consultation with a Lived Experience Advisory Panel and an academic panel. The review protocol was pre-registered (Prospero CRD42018090188).8951 titles, 366 abstracts and 121 full-text articles published January 2000-July 2018 were screened, of which 45 studies analysing 629 recovery narratives were included. A conceptual framework of mental health recovery narratives was developed, comprising nine dimensions , each containing between two and six types. Subgroup analysis indicated all dimensions were present across most subgroups, with Turning Points particularly evident in trauma-related studies.Recovery narratives are diverse and multidimensional. They may be non-linear and reject coherence. To a greater extent than illness narratives, they incorporate social, political and rights aspects. Approaches to supporting development of recovery narratives should expand rather than reduce available choices. Research into the narratives of more diverse populations is needed. The review supports trauma-informed approaches, and highlights the need to understand and support post-traumatic growth for people experiencing mental health issues. Recovery has become the established orientation within mental health policy and provision at national  and inteKnowledge about recovery is based primarily on the individual stories and resulting insights of those with lived experience of psychological distress . The shaSharing experiences through stories has become a central practice within recovery-based healthcare . Story-sstories and narratives are often used interchangeably, but following Smith and Sparkes [stories to refer to the actual tales people tell, and narratives when considering the dimensions and properties comprising particular stories, for example within the research context. Research interest in narratives grew rapidly in the late twentieth-century ‘narrative turn’ within the social sciences [The terms  Sparkes  the currsciences . Narratisciences .Narratives can be differentiated into internal narratives, told to oneself in response to events, and externally expressed narratives, told for and perhaps elicited by others . The focWithin health research, interest in personal narratives has translated into the investigation of ‘illness narratives’ as a key source of understanding the experiences of people with chronic conditions, including cancer , HIV andTypes of illness narrative have been researched, for example Frank’s differentiation of restitution, chaos and quest narratives . These tWithin mental health-related research, narrative approaches have equated mental illness with the breakdown of an individual’s coherent life story, whether as an inherent aspect of mental illness , or as aTherapists working within psychodynamic approaches may view listening to narratives as a way of gaining access to other areas of experience, such as relationship themes or other unconscious content . These aSocial constructionists adopt what may be a more emancipatory perspective, viewing narratives themselves as the central focus. A sense of identity, in this approach, is not discovered in but actively constructed by the act of telling stories , with thIn the 1990s, mental health research studies began to focus specifically on the recovery aspects of illness narratives . InflueStudies using narratives as a means of accessing other information take a broadly thematic approach. These studies are concerned with the content of participants’ narratives, and what can be learned about recovery from them  to review published documents presenting typologies or characteristics of mental health recovery narratives and (b) to use a modified narrative synthesis to develop a conceptual framework for the characterisation of mental health recovery narratives.A systematic review of the literature was carried out following PRISMA guidance . StudiesThe review was conducted as part of the Narrative Experiences Online (NEON) Programme, and the systematic review protocol was registered with PROSPERO Based on empirical data.Exclusion criteria were:Presents themes arising from narrative data without discussion of the characteristics of narratives themselvesNot primarily or partially about mental health, e.g. recovery from chronic pain, physical illness or drug/alcohol addictionBased on third-person accounts, e.g. stories told by family, friends, carers, professionalsFull text not available in English.A scoping search was undertaken and 12 publications were found. These informed the definition of mental health recovery narratives adopted by the current review (see above) and provided an overview of the nature of studies characterising mental health recovery narratives. The scoping search also informed the search strategy and confirmed that no systematic review had already been carried out in this area. Six search strategies were then selected to identify relevant publications.The electronic database search strategy was developed and piloted in consultation with two research librarians with expertise in systematic reviews. Databases were selected by conducting preliminary searches to gauge relevance of results and degree of overlap with other databases; by including those indexing the journals of key publications found in the scoping search; and by expert consultation. Due to the cross-disciplinary nature of narrative research, databases from health sciences, social sciences and the arts and humanities were searched.th July 2018: Applied and Complementary Medicine Database (AMED) accessed via OVID; Applied Social Science Index and Abstracts (ASSIA); Association for Computing Machinery Digital Library (ACM); Cumulative Index of Nursing and Applied Health Literature (CINAHL) via EBESCO; EMBASE; JSTOR; Linguistics and Language Behavior Abstracts (LLBA); Modern Languages Association International Bibliography (MLA) and Published International Literature on Traumatic Stress (PILOTS) Database both via ProQuest; MEDLINE; PsycINFO; Scopus via Elsevier; Arts and Humanities Citation Index and Social Science Citation Index, both via Web of Science.Fourteen bibliographic databases were searched from inception to 27A combination of subject heading and keyword searches was trialled to ensure sufficient specificity was maintained, while maximising the sensitivity of the search. MEDLINE was selected as the pilot database, as one of two databases which abstract the largest number of healthcare journals in the world . The folMental Disorders/Behavior/Psychological Phenomena/Mental Health/.ti,ab.1 or 2 or 3 or 4 or 5Mental Health Recovery/Psychiatric Rehabilitation/Resilience, Psychological/Hope/Quality of life/(Recover* or transform* or resilien* or surviv* or thriv* or endur* or rebuild* or hope* or conquer* or reclaim*).ti,ab.7 or 8 or 9 or 10 or 11 or 12Personal Narratives/Narration/Narrative therapy/.ti,ab.14 or 15 or 16(typol* or classif* or genre* or theme* or structur* or categor* or framework* or dimension* or format*).ti,ab.6 and 13 and 18 and 19.Keyword and, where applicable, subject heading searches were subsequently tailored to each database.st January 2000–27th July 2018: BMJ Medical Humanities; International Journal of Narrative Therapy and Community Work; Journal of Medical Humanities; Anthropology and Medicine; Qualitative Health Research.The tables of contents of five journals were hand-searched from 1Journals were selected by expert consultation and by including those featuring two or more included papers from the original electronic data search. The start date was selected as the year before two prominent early papers on recovery narratives identified in the scoping search were published , 47.http://www.researchintorecovery.com/RoR-conference-archive) and ENMESH (http://www.enmesh.eu/Enmesh_Conferences.html).Grey literature searches were conducted using Ethos, BASE and OpenGrey. Conference searches were undertaken using programmes available online from two recovery-oriented conferences: Refocus on Recovery (https://www.scottishrecovery.net) and Boston University Repository of Recovery Resources (https://cpr.bu.edu/resources/recovery-repository). Due to the large number of results found on Google Scholar  only the first ten pages of results were searched.Web-based searches were conducted using Google Scholar, ResearchGate and Academia.edu, and by searching the recovery-oriented websites Scottish Recovery Network  and Google Scholar (n = 2082).Papers identified by the search were uploaded to Endnote, and duplicates removed. Titles were screened for relevance against the inclusion criteria by the lead researcher (JLB), with a randomly-selected sample of 10% double-screened for inclusion by a second researcher (SRE) to establish a pre-defined adequate concordance of 90% or above.Potentially relevant abstracts were subsequently screened by the lead researcher (JLB). A sample of 20% of these abstracts was double-screened for inclusion by a second researcher (SRE). Full text was obtained for potentially relevant papers and eligibility decided by the lead researcher, with reasons for those excluded at full text retrieval stage documented in a PRISMA flow diagram.A data abstraction table was designed, and is presented in the supplementary material .There is little consensus regarding the most appropriate way of evaluating qualitative evidence within systematic reviews . HoweverAll studies published in academic journals were assessed for quality by two researchers (JLB and SRE). Qualitative studies were assessed using the Critical Appraisal Skills Programme qualitative checklist (CASP 2017), using thresholds modified from Butler and colleagues . The MixA three-stage narrative synthesis approach was used, modified from guidance for the conduct of narrative synthesis within systematic reviews . Principto preserve study authors’ terminology in naming dimensions and types as much as possible, while maintaining clarity and avoiding potentially non-inclusive language. Authors’ original terms are preserved in the tables presenting sources of information for the synthesis.to avoid over-combining so as not to risk collapsing discrete concepts which might remain useful separatelyA preliminary synthesis of studies using qualitative or mixed methods was developed by the lead researcher (JLB). Data were tabulated and analysed thematically to identify potential conceptual overlaps and/or similarities of language used to describe differing phenomena. Emerging dimensions fitted a framework commonly used in narrative and literary theory which considers narratives at three levels of form, structure and content (see for example ). These Quantitative data, such as length of narratives or linguistic categories within narratives, did not form part of the narrative synthesis and are presented in summary form in the supplementary material :.Relationships within and across studies were examined by the lead researcher (JLB), and subgroups of interest were identified. Publications within these subgroups were thematically analysed separately. Emergent themes were compared with the preliminary synthesis in order to identify areas of differing emphases and to assess robustness.The robustness of the preliminary synthesis was assessed using the following methods: subgroup analysis of studies rated as moderate and high quality; subgroup analysis of studies where narrator(s) were involved in the analysis process ; consultation with members of the NEON study Lived Experience Advisory Panel (LEAP) including people who have published their own mental health recovery narratives; consultation with an academic panel; and ongoing discussion and critical reflection by the research team. The synthesis was modified in response to findings; for example academic panel consultation strengthened internal coherence and LEAP consultation resulted in improved clarity of language used in the presentation of the synthesis and the definitions of dimensions and types.Forty-five publications were included in the review. Characteristics of included publications are presented in the supplementary material , and theThe 45 publications comprise qualitative (n = 41) and mixed methods studies (n = 4). Studies were conducted by research teams based in 11 countries: the UK (n = 16), the USA (n = 16), New Zealand (n = 3), Australia (n = 2), Canada (n = 2), one each from Chile, Germany, Greece and Israel, and two from multinational teams . Publication was between 1997 and 2018. Fifteen of the 45 were published between 2006 and 2008.Thirty-one publications explicitly use the term ‘recovery narrative’ or close variants to describe their source data. Variants include “stories of healing”, “redemption narratives” and “narrative re-storying”. This group includes a paper which contains, to the current authors’ knowledge, the earliest naming of ‘recovery narratives’ as a phenomenon distinct from ‘illness narratives’ . FourteeThe 29 studies published in academic journals were assessed. Of the 25 qualitative studies, two (8%) were evaluated as high-quality, 10 (40%) as moderate-quality and 13 (52%) as low-quality. Of the four mixed-methods studies, two (50%) were evaluated as high quality, one (25%) as moderate and one (25%) as low quality. Sixteen studies were excluded from quality assessment, comprising doctoral theses (n = 11), reports (n = 3) and books or book chapters (n = 2).The 45 included publications analysed 629 first-person lived experience accounts. Gender of narrators was 59% (n = 370) female, 34% (n = 215) male and 7% (n = 44) not stated. Ages ranged from 8 to 79 years old, with 43 (96%) of publications only including adult (18 years or older) narrators. 40% of narrators were identified as white and 17% as BAME, while the ethnic identity of 43% of narrators could not be identified, as 26 of the 45 publications (58%) did not provide breakdowns of ethnicity. Narrators had experience of conditions or circumstances from across the spectrum of mental ill health. Those named (either by the research team or self-identified by narrators) were: anorexia, anxiety, bipolar disorder, borderline personality disorder, bulimia, childhood maltreatment, co-existing mental health and substance use issues, depression, dissociative identity disorder, eating disorders, mania, manic depression, nervous breakdown, personality disorders, psychosis, PTSD, schizophrenia, social anxiety, survival of sexual abuse and voice-hearing.Nine dimensions were derived from thematic analysis of included publications, with each dimension containing a number of types. Types are not presented as discrete; some included authors state that more than one may be present within a narrative. The final synthesis is presented as Four dimensions related to narrative form were identified: Genre, Positioning, Tone and Relationship with Recovery. The central question common to all four dimensions is ‘what kind of story is this?’Twelve publications identified different genres of mental health recovery narrative, also using the following synonyms for genre: narrative type, form, plot and theme. Four types were synthesised from the 20 genres presented, using terms adapted from Thornhill and colleagues’ study . These aAll 12 publications sought to identify genres in order to examine constructions of meaning by narrators. Nine publications (75%) cited Frank’s genres of restitution, chaos and quest illness narratives  as eitheThe ‘Escape’ type comprises narratives of escape from and resistance to abuse, threat, stigma and persecution. Images of entrapment and/or of a fight for survival may be used . Escape The ‘Endurance’ type comprises narratives of loss, trauma, difficult circumstances and/or seemingly insurmountable odds. They may employ images of weathering storms or battening down the hatches to conserve energy . They maThe ‘Endeavour’ type comprises narratives incorporating positive aspects, coping strategies and/or plans, and an acceptance of difficulties as an ongoing factor of recovery. Narrators may feel they are active agents of change , or theyThe ‘Enlightenment’ type comprises narratives of transformation. The narrator views the illness/trauma as ultimately positive, as new perspectives have been gained from it. They may describe recovery as a journey of exploration or discovery , leadingEleven publications identified ways in which mental health recovery narratives can be positioned in relation to the clinical model of mental health. The following synonyms for positioning were also used: major theme/plot, typologies of narratives genre, and narrative types. Unlike those in Genre, these publications specifically foreground social and political considerations of the mental health system in the identification of narrative types. The ‘mental health system’ is defined for the purposes of this paper as being the dominant clinical mental health provision of the country involved. Three types were synthesised from the 13 positions presented, using terms identified by the current research team. These are shown in ‘Recovery within the system’ comprises narratives incorporating positive experiences of clinical mental health services. Diagnosis may be experienced as empowering, and treatment, services and/or relationships with practitioners as enabling, positive or a salvation.‘Recovery despite the system’ comprises narratives of protest, in opposition to the clinical model of mental illness and/or mental health services and systems. They may incorporate experiences of maltreatment by mental health services. They may seek to recover the narrator’s own voice, sense of agency and purpose , and may‘Recovery outside of the system’ comprises narratives in which clinical mental health services do not feature, or feature only minimally. They may not engage with psychiatric definitions or psychological concepts of personal growth; presenting experiences of living a “good life” beyond a focus on individual factors . They maThree publications identified different emotional tones present within mental health recovery narratives, with one using the term “self-positions” . Six typNine publications identified different ways of relating to the concept of recovery within mental health recovery narratives. The following synonyms were also used: recovery talk, narrative positions or types, framings of recovery, narrative motifs, core narratives and narrative genres. Four types were synthesised from the 15 presented, using terms adapted from Barnett and Lapsley’s typology . These aThe ‘Recovered’ type comprises narratives presenting recovery as an outcome which has been achieved. Narrators see the illness or distress as being in the past . There mThe ‘Living well’ type comprises narratives presenting recovery as a process within which the narrator is well-established. Narrators are living well in the presence or absence of mental illness or distress , and seeThe ‘Making progress’ type comprises narratives presenting recovery as a process in which they are seeing some progress. Narrators present confidence in their ability to cope despite feeling relatively close to the disruptions of mental distress or trauma .The ‘Surviving day to day’ type comprises narratives presenting recovery as a journey on which the narrator is tentatively engaging. The narrator may be in a new, difficult or dangerous situation  where itThree dimensions related to narrative structure were identified: Narrative Trajectory, Use of Turning Points and Narrative Sequence. The central question common to all three is ‘what shape does this story take?’Seven publications identified different trajectories present within mental health recovery narratives, also using the following descriptions: types of emotional distress, narrative shapes, plots and structures. These may describe the shape of whole narratives or of sequences within narratives. Four types were synthesised from the 14 presented, shown in The ‘Upward spiral’ type comprises narratives describing a journey with an overall ascending progression toward recovery. They may be described as narratives of revelation or purposeful suffering , or evolThe ‘Up and down’ type comprises narratives describing a non-linear journey which challenges the progressive trajectory of spiralling ever forward towards health . They coThe ‘Horizontal’ type comprises narratives without significant upturns or downturns. The narrator may feel that they are stagnating , or takiThe ‘Interrupted’ type comprises narratives describing a journey interrupted by an unexpected crisis or difficulty, after which the narrator’s life has returned to its prior state .Eleven publications identified turning points as being a characteristic of mental health recovery narratives, but defined ‘turning points’ in three different ways. Three types were synthesized, shown in The ‘Re-storying’ type comprises narratives within which turning points are considered as moments in which a narrator gains a new understanding of their experiences . It may The ‘Change for the better’ type comprises narratives within which turning points are considered as moments of transition, followed by sequences leading to improvement  or positThe ‘Change for the better or worse’ type comprises narratives within which turning points are considered as critical life events  or signiEight publications identified different sequences within mental health recovery narratives, also using the following terms: stages, narrative shape, typologies and structure. Eight types were synthesised from the 37 sequences described. These are shown in Two dimensions related to narrative content were identified: Protagonists and Use of Metaphor. The central question common to them both is ‘what resources have been deployed in the telling of this story?’Four publications identified different protagonists within mental health recovery narrative, also using the following synonyms: narrators, biographical types, archetypal protagonists, major players and heroes/supporting cast. Three types were synthesised from the 15 protagonists presented, shown in ‘Personal factors’ are the micro-level or inter/intra-personal factors within a mental health recovery narrative. Most commonly the narrator him or herself, who may be characterised  or posit‘Socio-cultural factors’ are the meso-level factors within a mental health recovery narrative, including family and friendship dynamics, groups or local organisations, mental health staff and services. These may be “supporters or villains”, exerting positive or negative effects on the narrative .‘Systemic factors’ are macro-level factors within a mental health recovery narrative, named in included publications as the wider community , and theThree publications focused on the use of metaphor within mental health recovery narrative. Two types were synthesised from the six presented. These are shown in ‘Distress metaphors’ may depict a deep descent of the self, a “spiralling out of control” . They ma‘Recovery metaphors’ may depict health as the main road to which one must return . They maFour subgroup analyses were undertaken of papers published in academic journals. Analysis of moderate and high-quality papers  found that the following items from the conceptual framework were not present: two dimensions (Protagonists and Use of Metaphor); one type of Genre (‘Endeavour’); two types of Emotional Tone (‘Buoyant’ and ‘Tragic’), and a type within Relationship with Recovery (‘Recovered’). Within the Narrative Sequence dimension three types were found .Analysis of papers with narrator involvement at analysis stage  found that eight of the nine dimensions and all corresponding types were present. The Emotional Tone dimension and its types were not found in these papers. Within the Narrative Sequence dimension eight more detailed types were found .Analysis of papers focusing exclusively on narratives of psychosis (n = 12) found no significant differences of emphases when compared with papers focusing on other conditions.Subgroup analysis found that all papers focusing on trauma (n = 6) focused on dimensions within the superordinate category of Narrative Structure, namely Trajectory, Use of Turning Points and Narrative Sequence. All six discussed Use of Turning Points. One also included discussion of Genre .This review has identified the existence of a sizeable body of qualitative and mixed-methods literature describing the multidimensional ways in which mental health recovery narratives have been characterised. Forty-five publications were identified by the systematic review search. The literature was multidisciplinary and published in a wide range of journals, spanning 21 years of research. The 45 papers represent analysis of a total of 629 first-person lived experience accounts of psychological distress and recovery, from narrators representing many demographics. A key contribution of this review is to collate and synthesise these disparate narratives. This provides an accessible resource for practitioners, researchers and others interested in the distinctly heterogeneous ways in which people both recover and narrate their recovery from psychological distress and/or trauma. It will also inform a future trial of the impact of accessing recovery narratives online on the quality of life of people experiencing psychosis (ISRCTN11152837).The narrative synthesis then moves beyond this work by providing a more comprehensive framework for characterising narratives than any single model can offer. The synthesis found that mental health recovery narratives can be characterised under three superordinate categories of narrative form, structure and content, and nine dimensions: Genre, Positioning, Emotional Tone, Relationship with Recovery, Trajectory, Use of Turning Points, Narrative Sequence, Protagonists and Use of Metaphor. This extends previous work such as that of Frank , 24 and A key finding of this review is that none of the included publications claim a ‘right way’ to narrate recovery or to characterise mental health recovery narratives. Care is taken by authors of included publications to stipulate that narrators speak from a wide range of circumstances, and that no type of recovery narrative should be pathologised or, by implication, valorised, in relation to others .Narrators do not present single or static types of narratives. As with “small stories” or exchanges in everyday life , narratois, but what its possibilities are—what it can be. Narrators express their recovery through many types and shapes of story, drawing on a variety of resources to do so. This is appropriate for a recovery paradigm wherein meaning-making is a highly individual process [Correspondingly, the current conceptual framework is not presented as a definitive or exhaustive list of types; but as a “network or ‘plane’ of linked concepts that together provide a comprehensive understanding of a phenomenon” ( p.51). F process . It echo process  p. 5).Mental health recovery narratives express individual meaning-making processes within recovery; they also highlight wider socio-economic and systemic influences. Narrators in the included publications populate their recovery narratives with supportive and hindering factors at individual, socio-cultural and systemic levels. This finding supports research presenting recovery as a multidimensional process, involving biomedical, psychological, social and socio-political resources and components . It respRecovery narrative analysis in the included publications is rooted in influential work within the medical humanities on illness narratives, notably by psychiatrist and anthropologist Arthur Kleinman  and mediRecovery narratives have been characterised by genre, as have illness narratives, but add a new type, first identified by Thornhill and colleagues: narratives of “Escape” . The preRecovery narratives are additionally characterised by consideration of narrative positioning in relation to a clinical model of mental health. Positioning relates to the “social and emotional stances that individuals take vis á vis real or imagined others” .Frank’s  restitutMental health recovery narratives also challenge the clinical model more directly, providing evidence and mirroring research that recovery can happen without the intervention of services . NarratiNarratives opposing or outside of the mental health system have played a crucial role in survivor activism, and ultimately in establishing recovery as an orientation within services. Given this historical context, narratives endorsing mental health treatments or services may be dismissed as conforming to the dominant clinical model, rather than expressing authentic experience–the narrator perhaps performing the role of the ‘good patient’. The potential for narrative conformity in particular groups has been researched, including Frank’s own exploration of Alcoholics Anonymous groups . HoweverFrank  defines Firstly, the review includes narratives (synthesised as ‘Endurance’ narratives) which present recovery as being possible in the midst of considerable chaos (see for example ). ChaotiSecondly, subgroup analysis of studies focusing on narratives of psychosis found no difference in characteristics or emphasis when compared with narratives of other forms of psychological distress. Insofar as psychosis involves chaos, it is noteworthy that people experiencing psychosis narrate recovery with the same characteristic features and multi-dimensional meanings as other narratives. This finding challenges the stereotype of people experiencing psychosis as being incompetent or incapable of insight into their experiences. It supports moves to broaden the concept of ‘insight’ within psychosis services to incorporate the ‘narrative insight’  or explaFrank’s quest narratives , synthesA core domain of recovery is its non-linearity . This isIn contrast, the Narrative Sequence dimension comprises surprisingly linear progressions through eight sequences, from experiences of distress/trauma through a turning point to recovery. Following DiClemente and Prochaska’s lead with their transtheoretical model of change , it woulAn important contribution of the current synthesis is to highlight the kinds of recovery narratives, narrators or other factors missing from research to date, and some significant gaps were noted. One such gap was that only one of the included publications provided a clear definition of mental health recovery narratives .All included publications except one focused on the narratives of individuals. The exception was Anderson and Hiersteiner’s study which used group interviews to construct a group narrative of recovery-survival of childhood sexual abuse . FurtherAll narratives in their original formats were either written or spoken, and none was accessed online. Two publications complemented the collection of written/spoken narratives by additionally undertaking art and music-based interventions , 91. WorSome included publications focus on “exemplar” narratives or “seminal accounts”, for example , 56, 79.Finally, although the presence of socio-cultural and systemic-level factors in recovery were noted by two studies , 96, repThe comprehensive search strategy is one strength of this review, reflecting multidisciplinary interest in narrative approaches. Another is the quality assessment process. In addition to standard critical assessment of methodological quality, a subgroup analysis was carried out on studies incorporating narrator validation or co-production, as a complementary measure of trustworthiness. This is in line with the principles of agency and giving voice to potentially marginalised groups which are central to both recovery and narrative paradigms . It is nThe robustness of the synthesis is another strength. Subgroup analyses of studies of high methodological quality and of high trustworthiness validated the findings of the preliminary synthesis. Expert consultation was carried out with academic experts, which strengthened the internal coherence of the synthesis, and a Lived Experience Advisory Panel, which increased the trustworthiness and ecological validity of the synthesis.A further strength of this review is that its first and second authors have dual identities as both researchers and people with lived experience of mental ill health and recovery. This informed in particular the principles adopted in the development of the narrative synthesis.The review is limited by its search for publications available in English only. This led to included publications which in the main reflect criticism that research into recovery is monocultural . It is lA second limitation is that the involvement of an external Lived Experience Advisory Panel was consultative. The emergence of models of collaborative data analysis  can suppThe reviewed publications include narratives across the range of conditions and experiences of psychological distress. Subgroup analysis of psychosis and of trauma studies found no important differences in their characterisation compared with those narrating other conditions. This resonates with research on transdiagnostic approaches , which rThe framework provided by the current synthesis could form the basis for an inventory of recovery narrative characteristics. This could be used by those delivering narrative-based interventions as an evidence-based template, ensuring diversity in two ways.Firstly, such an inventory could be used as a basis for expanding the range of examples and resources offered within online, written and course-based guides to developing recovery stories. Narrative-based courses could ensure that their content and the way they are structured do not imply that particular elements must be present in order for the narrative to be a recovery story. Similarly, repositories collating stories as a resource may choose to use the framework as a tool to avoid potential biases. Requiring an emphasis, for example, on personal choice and responsibility within stories as criteria for inclusion may support the criticism that the use of narratives by mental health services has been co-opted in the service of neo-liberal agendas .Secondly, the framework could be used to identify potential gaps in collections, highlighting areas where effort may need to be made to target specific communities to ensure their narratives are also included. This would provide a diversity metric for recovery story collections.It is noted that the three higher-methodological quality papers which focused on narrative sequence identified a three-stage sequence only, wherein the first stage is a variation on ‘traumatic past’. This contrasts with other publications which include a sequence named ‘life before problems begin’ or similar. Considerable work is currently being undertaken on the overlap between childhood trauma and experiences of mental ill health , 136, anFinally, the current synthesis may form the basis for a matching tool within narrative interventions. This may contribute towards increasing the confidence of practitioners in connecting individuals with the kinds of recovery narratives likely to be most beneficial to them within a particular context or time. Mental health practitioners, and recovery narratives themselves, can play a crucial role as “holders of hope” at times when an individual may be unable to construct, hold onto or believe in their own narratives of recovery . HoweverThis review extends the literature on mental health recovery narratives, by synthesising the various ways in which they have been characterized to produce a conceptual framework. Recovery narratives have played a vital role in establishing the recovery paradigm for survivor movements and individuals, and within mental health services and policy. Care is needed to ensure that recovery narrative interventions are used to expand the available choices within the narrating of recovery instead of curtailing them. Practitioners using narrative interventions will need to consider possible limitations of the form, structure and content of any tools offered. Mental health narrative researchers should aim to increase the diversity of populations invited to tell their recovery story.S1 FileReview protocol registered with PROSPERO .(PDF)Click here for additional data file.S1 TableShowing characteristics and findings of the 45 included papers.(XLSX)Click here for additional data file.S2 TableShowing a summary of quantitative findings from two papers.(XLSX)Click here for additional data file.S3 TableShowing the page numbers on which Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) are reported.(DOC)Click here for additional data file.


The family of ribosomal proteins S1 contains about 20% of all bacterial proteins including the S1 domain. An important feature of this family is multiple copies of structural domains in bacteria, the number of which changes in a strictly limited range from one to six. In this study, the automated exhaustive analysis of 1453 sequences of S1 allowed us to demonstrate that the number of domains in S1 is a distinctive characteristic for phylogenetic bacterial grouping in main phyla. 1453 sequences of S1 were identified in 25 out of 30 different phyla according to the List of Prokaryotic Names with Standing in Nomenclature. About 62% of all records are identified as six-domain S1 proteins, which belong to phylum Proteobacteria. Four-domain S1 are identified mainly in proteins from phylum Firmicutes and Actinobacteria. Records belonging to these phyla are 33% of all records. The least represented two-domain S1 are about 0.6% of all records. The third and fourth domains for the most representative four- and six-domain S1 have the highest percentage of identity with the S1 domain from polynucleotide phosphorylase and S1 domains from one-domain S1. In addition, for these groups, the central part of S1 (the third domain) is more conserved than the terminal domains. A comprehensive investigation of the distribution of ribosomal proteins and finding of the specific signatures of ribosomal evolution between and within the ribosomal protein domains is an actual task, which provides new insights into the emergence and evolution of the protein component of ribosomes –4.in vivo and interact with the mRNA-like part of the tmRNA molecule [Escherichia coli, four unique repeats were found [E. coli) [As demonstrated in our recent paper , the fammolecule ,9. Like molecule . In addimolecule ,12. Initre found . This reE. coli) . 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The alignments of S1 sequences with the S1 domain from polynucleotide phosphorylase (PNPase) and other proteins S1 containing one structural domain allowed us to find the sequential number of the domain with the highest value of identity and the largest number of representatives. In addition, we showed that the central part for some groups of S1 (proteins with five and six domains) is more conserved than the terminal parts.The automated advanced exhaustive analysis allowed us to choose 1453 records corresponding to search parameters. As noted above see , for eachttp://www.bacterio.net/). Some phyla include only several types of bacteria , while about 90–95% of all known bacteria are included in such phyla as Proteobacteria, Bacteroidetes, Cyanobacteria, Actinobacteria and Firmicutes. The 1453 S1 sequences were identified in 25 different phyla (except candidate phyla). All studied phyla of bacteria and the number of S1 domains found in them are shown in the sunburst chart , st chart .E. coli [Mycoplasma auris– 110 amino acid residues). One S1 domain is also found in very few bacteria of phylum Actinobacteria. One S1 domain is also identified in some bacteria of phyla Firmicutes, Proteobacteria, and Bacteroidetes. Interestingly, in all studied phyla only several bacteria (0.6%) containing two S1 domains were found . In all cases, Cyanobacteria with an average length of ribosomal protein S1 of about 350 amino acid residues have three S1 domains; also, some representatives of phyla Firmicutes, Actinobacteria, and Proteobacteria have three-domain S1 proteins. Generally, three-domain S1 proteins are identified in 1.9% cases. Records with four S1 domains were identified in 33% cases from all investigated ribosomal proteins S1. Almost all analyzed bacteria (with the protein length of 390 amino acid residues) in this group relate to phyla Actinobacteria  and Firmicutes . In bacteria of the monotypic (consisting of one Deinococci class) phylum Deinococcus-Thermus, the length of protein S1 is on average about 530 amino acid residues, and these bacteria have always five S1 domains . Five S1 domains are also found in bacteria of phyla Thermotogae, Synergistetes, Haloplasmatales. Generally, five-domain S1 proteins compose 1.1% from all investigated ribosomal proteins S1. About 62% of the records are identified as proteins containing six S1 domains. Generally, these proteins belong to phylum Proteobacteria. Ribosomal proteins S1 from bacteria of the phylum Chlorobi (green sulfur bacteria) also have six S1 domains. Gram-negative bacteria containing six S1 domains include Spirochaetes, Bacteroidetes, Chlamidia, and Proteobacteria . In these bacteria, the length of the ribosomal protein S1 averages about 570 amino acid residues. Gram-positive bacteria contain different numbers of the S1 domain depending on the phylum. Phylogenetic bacterial grouping according to the number of structural domains and length considering 1453 S1 sequences is shown in Only 0.8% from all investigated ribosomal proteins S1 contain one S1 domain. The most represented in this group is the phylum Tenericutes. It should be noted that mycoplasma is the simplest independent reproducing living organism. The total amount of its genetic information is four times less than that of E. coli . The shoE.coli, the biochemical experimental study of various fragments allowed to establish the functions of individual protein domains and parts. It was shown that the removal of one S1 domain from the C-terminus or two S1 domains from the N-terminus of a protein decreases only the effectiveness of the protein functions, but not its functional capabilities [The automated advanced exhaustive analysis of 1453 S1 sequences allowed us to demonstrate that the number of structural domains in S1 is a distinctive feature  for the phylogenetic grouping of bacteria in the main phyla. Several attempts have been made to classify ribosomal protein S1 according to a different number of sequences. We are the first who has performed an exhaustive analysis of S1. As noted above, S1 is identified in 25 different phyla. 13 phyla were studied by Salah et al.  They usebilities ,22. Wherbilities .http://bioinfo.protres.ru/other/Amount_and_borders_S1_domain.xlsx) demonstrate that the ribosomal protein S1 in this group always contains six S1 domains  from  strands . This OB strands .Alignments of 1453 S1 sequences and the S1 domain of PNPase allowed us to calculate the average identity for each S1 domains and find the sequence number (No.) of the domain with the high value of identity with the RNA-binding S1 domain of PNPase  with theIn addition, for the four- and six-domain S1 proteins, the fourth domains are also have a high value of identity with PNPase and a large number of representatives. It should be noted that the residues Phe19, Phe22, His34, Asp64, and Arg68  (in someM. auris (the shortest full length S1 protein with 110 amino acid residues) allow us to find the domain with the highest value of identity and the largest number of representatives located in the third domain (M. auris and the number of representatives were found for the fourth domain (for four-domain proteins: 220 records with 54% identity and for six-containing domains: 634 records with 49% identity). The same correlation  is found when the S1 protein sequences are aligned with other one-domain S1 proteins .Alignments of the S1 sequences and the S1 domain protein from d domain . For fouE.coli) and S1 domains from one-domain S1 proteins  are the third and fourth domains for the most representative four- and six-domain S1 proteins.Thus, the most conserved domains with the S1 domain from PNPase  using the Pairwise2 module from BioPython. The percentages of identity for these domains were calculated using standard parameters. The maximal and minimal values of identity for each group are marked in http://bioinfo.protres.ru/other/multiple_alignment.xlsx) using the Clustal Omega service . The obtained results are also shown in To check the equivalence of domain characteristics, each S1 domain in the corresponding S1 protein sequence in different groups (according to the domain number) was aligned in pairs (It is noteworthy that S1 proteins containing one-domain have a low percentage of identity among themselves (27%), as well as in one phylum (Tenericutes). Nevertheless, the predicted secondary structures for these sequences  are veryThe first and the second domains of S1 proteins containing two structural domains have 38% identity, and pairs with maximum and minimal values of identity are identified for the remaining domains. The first and second domains in S1 proteins containing two structural domains also have a low percentage of identity in the domains: 27% and 30%, respectively.For S1 proteins containing three structural domains, the maximal value of identity is revealed between the first and third domains (53%) and the minimal value between the first and the second domains (42%). Wherein, the third domain has the maximal percentage of identity (57%) among other domains for this group.For S1 proteins containing four structural domains, the maximal value of identity is revealed between the third and fourth domains (78%) and the minimal value of identity between the second and third domains. The third domain has also the maximal percentage of homology (66%) among other domains of this group.The third and fourth domains in the group of S1 proteins containing five structural domains have the maximal percentage of identity (66%), while the second and fifth domains have the lowest percentage of identity (43%). In this group, the fourth domain has the maximal percentage of identity among other domains (49%).For the most represented S1 proteins containing six structural domains, as well as for S1 proteins with four and five domains, the maximal values of identity are identified between the third and fourth domains (71%) and the minimal values between the first and second (39%). It should be noted that the third and fourth domains  also have the maximal values of identity with the S1 domain from PNPase and S1 protein sequences with different single-domain S1 proteins. Moreover, the third domain has the maximal percentage of identity among other domains in this group. Thus, the obtained results showed that for long S1 proteins (five- and six-domain ones) the central part of the proteins (the third domain) is more conserved than the terminal domains.The problem of understanding the nature of protein repeats, the corresponding functions for each repeat, and their evolution is still unclear. These repeats evolved from a common ancestor, which necessarily contained a single repeat . Some auE.coli) and S1 domains from one-domain S1 proteins   is sufficient to perform all necessary functions. As shown above, the third domain in this group has the maximal identity (68%) among other domains. In addition, this domain has the highest identity with the S1 domain from PNPase (licutes) .As can be seen from E.coli. It was shown that cutting one S1 domain from the C-terminus or two S1 domains from the N-terminus of a protein decreases only the effectiveness of protein functions, but not its functional capabilities [This suggestion is consistent with experimental data. One of the well-studied proteins with six S1 domain repeats is the bacterial 30S ribosomal protein S1 from bilities ,22.Clostridium, Bacillus, Lactobacillus, and Streptococcus, a reasonable assumption was made regarding their evolutionary relationship with the division Firmicutes [It should be noted that the bacterial 30S ribosomal protein S1 from parasitic bacteria Mollicutes effectively performs the main functions of RNA-binding . There irmicutes ,34. A mormicutes . In turnIn the future, a more detailed analysis of the phylogenetic and evolutionary relationships between one-containing S1 domain proteins and the third and/or fourth domains will allow us to conclude the evolutionary development of the family of bacterial proteins S1 and verify our previous suggestion about the effect of reducing the evolution of the number of repeats in the family of 30S ribosomal proteins S1 .Studies of 1453 S1 sequences available in the UniProt database showed that the number of structural domains in the ribosomal proteins S1 can be considered as a distinctive feature for the phylogenetic grouping of bacteria in 25 different bacterial phyla. It can be assumed that bacteria affiliation may be associated with the structural features and multifunctional activity of ribosomal proteins S1. The obtained results differ from the data obtained for small data sets ,34, and Proteins belonging to the phylum Proteobacteria and containing six S1 domains are mainly presented. This fact undoubtedly is associated with the wide distribution of this phylum bacteria and the presence of their sequences in the UniProt database. However, the stability of the number of multiple structural domains in these bacteria is apparently an evolutionary feature, that is necessary for functional diversity. The least represented proteins contain two S1 domains. The sequences found in this group belong to bacteria of phyla Actinobacteria, Firmicutes, and Proteobacteria and are mainly represented by an individual representative in each bacterial class within the phylum.Verification of the equivalence of domain characteristics showed that for long S1 proteins (five- and six-domain S1 proteins), the central part of the proteins is more conserved than the terminal domains, and, apparently, is vital for the activity and functionality of S1. Moreover, when aligning sequences between individual domains in each group, a rather low percentage of identity is revealed, which indicate that for the general functioning of these proteins the structure scaffold (OB-fold) is obviously more important than the amino acid sequence.Based on the obtained data, further investigations of possible evolutionary, functional, and structural relationships between bacterial phyla and bacterial classes within each phylum will reveal the relationship between the number of structural repeats and the specificity of the multifunctional activity of proteins of this family. Besides, the study of evolutionary relationships for the considered phyla will allow us to find evidence for one of the proposed theories of the evolutionary development of proteins with structural repeats: from multiple assemblies to single or vice versa.«30s ribosomal protein s1», «ribosomal protein s1», «30s ribosomal protein s1 (ec 1.17.1.2)», «30s ribosomal protein s1 », «ribosomal protein s1 domain protein», «rna binding protein s1», «rna binding s1 domain protein», «s1 rna binding domain protein» in the protein name were selected (UniProt release 2018_04). Then the obtained array of data was used to choose only proteins encoded by the rpsA gene or its analog, for example, rpsA_1, rpsA_2, rpsA_3 etc. Only this gene, coding the ribosomal protein S1, in the European nucleotide archive  is affiliated to the STD class, i.e. the class of standard annotated sequences. Therefore, the selection of records for the rpsA gene made it possible to regard the obtained collection as reliable, complete and sufficient for the aim of the study. From the obtained dataset records with six-digital identification numbers (annotated records in the UniProt database) were selected. All data were collected in one file that was the basis for further analysis, namely for collection of data on the number of structural domains and for phylogenetic grouping in the main bacterial phyla (http://bioinfo.protres.ru/other/uniprot_ids.xlsx). Records characterized by the presence of the word “candidate” were removed from our dataset, because there is not enough information for such records to call it a new species and define phylum according to the International Code of Nomenclature of Bacteria.To make a representative dataset of records for the family of ribosomal proteins S1 from the UniProt database, all records for the bacteria containing any one of the keywords http://www.ebi.ac.uk/interpro/).Four databases of protein domains, SMART (about 1200 domains), Pfam (16295 families of proteins united in 559 clans), PROSITE , and SUPFAM (1962 protein domains and 3245 different types of organisms) were analyzed in the study. The values of the number of domains S1 corresponding to the data from each database were selected for each analyzed record. If no data on the number of domains in one of the analyzed bases were available (None), this number was taken to be zero. Profiles of additional domains (not S1) according to their sequences were taken from the database InterPro (http://www.ebi.ac.uk/interpro) were also removed from the investigated dataset.The values of the number of S1 domains corresponding to the SMART database (about 1200 domains), were selected for each analyzed record. If no data on the number of domains in one of the analyzed bases were available (None), this number was taken to be zero (these records were removed from investigated dataset). Accurate borders for each S1 domain for each record were taken from the UniProt database . Records with additional domains (not S1) according to the InterPro database (http://www.ncbi.nlm.nih.gov/taxonomy)Bacteria were classified in main taxonomic categories  in accord with the Taxonomic database NCBI . Clustal Omega is a multiple sequence alignment program that uses seeded guide trees and HMM profile-profile techniques to generate alignments between three or more sequences. In our work standard parameters of this program were used.A global pairwise sequence alignment  using a dynamic programming algorithm was used. The Multiple Sequence Alignment was implemented by the Clustal Omega service (https://www.python.org/). Pairwise2 module from Biopython was used for the alignment functions to get global alignments between two sequences. Bio.pairwise2 uses the Smith-Waterman algorithm for local alignment, and Needleman-Wunsch for global alignment with standard parameters.Algorithms of search, collection, alignment, representation and analysis of the data were realized using the freely available programming language Python 3 (S1 Table(DOCX)Click here for additional data file.


Anti-EGFR therapy is used to treat metastatic colorectal cancer (CRC) patients, for which initial response rates of 10–20% have been achieved. Although the presence of HER2 amplifications and oncogenic mutations in KRAS, NRAS, and BRAF are associated with EGFR-targeted therapy resistance, for a large population of CRC patients the underlying mechanism of RAS-MEK-ERK hyperactivation is not clear. Loss-of-function mutations in RASGAPs are often speculated in literature to promote CRC growth as being negative regulators of RAS, but direct experimental evidence is lacking. We generated a CRISPR-mediated knock out panel of all RASGAPs in patient-derived CRC organoids and found that only loss of NF1, but no other RASGAPs e.g. RASA1, results in enhanced RAS-ERK signal amplification and improved tolerance towards limited EGF stimulation. Our data suggests that NF1-deficient CRCs are likely not responsive to anti-EGFR monotherapy and can potentially function as a biomarker for CRC progression. Colorectal cancer (CRC) is one of the most common cancers and the third leading cause of worldwide cancer deaths (IARC). The use of monoclonal antibodies (moAbs) targeting the epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab, together with chemotherapy has shown a clinical benefit for the treatment of patients with metastatic CRC (mCRC) –3. The bRAS proteins act as molecular switches that cycle between inactive GDP-bound and active GTP-bound states. Active GTP-bound RAS can stimulate a large variety of downstream signaling cascades, including the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways, to promote proliferation, migration and survival. The activation of RAS is tightly regulated by guanine nucleotide exchange factors (GEFs) and GTPases activating proteins (GAPs). GEFs accelerate the dissociation of GDP from RAS, whereas GAPs enhance the intrinsic rate of GTP hydrolysis of RAS . ActivatIn this regard, RASGAPs that act as negative regulators of RAS signaling are frequently implicated in tumorigenesis. In the human genome, ten functional RASGAP genes have been identified. Genetic analysis of tumor samples only identified a significant number of inactivating mutations in the RASGAPs neurofibromin (NF1) and RASA1 (p120GAP), suggesting that these two RASGAPs can function as tumor suppressors. Moreover, ongoing sequencing efforts of larger patient cohorts may increase the detection of low abundant loss-of-function mutations in several other RASGAPs , 25.NF1 are frequently associated with a large variety of cancers, such as melanoma [RASA1 gene have also been detected in a number of cancers, such as lung squamous carcinoma [Loss-of-function mutations in melanoma –29, leukmelanoma –32, gliomelanoma , and lunmelanoma . Moreovemelanoma –36, inclmelanoma  and lungmelanoma . Inactivarcinoma , stomacharcinoma , leukemiarcinoma , and heaarcinoma  cancer, In line with their molecular function, a suggestive tumor suppressive role for RASGAPs in CRC has been proposed based on association studies –46, as wHere, using CRISPR-mediated knock out lines in patient-derived CRC organoids that are otherwise wild type for the RAS pathway, we investigate the role of RASGAPs in CRC progression and in relation to EGFR signaling. Surprisingly, in contrast to widely accepted assumptions, but in line with overall mutation frequencies, we show that only the loss of NF1, but no other RASGAPs, can act as an amplifier of MAPK signaling. As such, NF1-deficiency contributes to CRC progression by minimizing its dependence on EGF-ligand stimulated MAPK signaling.RAS or BRAF.Strong activating mutations of RAS pathway effectors tend to occur in a mutually exclusive manner, most pronounced for oncogenic mutations in either NF1 tend to be mutual exclusive with activating mutations in RAS and BRAF (TCGA) in these tumors   Figure . Howeveractivity , suggestIn contrast to lung adenocarcinoma and melanoma patients, the numbers of inactivating mutations in colorectal adenocarcinoma patients are low in all RASGAP genes (TCGA), including NF1 Figure . For CRCThus, whereas mutual exclusivity of NF1-loss, in combination with previous experimental data , 36, 38,To circumvent the lack of patient-data regarding loss of RASGAP expression, we set out to test the function of RASGAPs in CRC in an experimental setting. For this we utilize a patient-derived tumor organoid (P18T) with loss-of-function mutations in the WNT (APC) and TP53 pathway, but that is wild type for the RAS pathway and as such requires EGF-mediated growth factor signaling for growth and survival . With thSurprisingly, we found that only the loss of NF1 GAP activity, but not of the other RASGAPs, resulted in a significant organoid growth upon intermittent EGFR inhibition Figure . In contNF1 and RASA1 in engineered tumor organoids that are also deficient in APC and TP53 (commonly referred to as tumor progression organoid 2 (TPO2)) [Since small sized organoids were observed for most conditions, including the negative control, we labelled all living organoids at the end point with calcein green to perform accurate measurements of number and size of organoids. Again, it illustrates that the number of organoids that are significantly larger in size is only observed after loss of NF1 activity in relation to the other RASGAP knock out organoids Figure . Importa (TPO2)) . ReassurTogether, these results indicate that only loss of NF1 activity promotes the outgrowth of CRCs upon limited EGFR signaling. The lack of participation by other RASGAPs is surprising, but consistent with the non-redundant and tissue-specific functions of RASGAPs .NF1 and RASA1 knock out lines in the CRC background of P18T   Figure  and 3D. ene body , accompaene body . Althougrganoids , we coule Figure . As we wFirst, we validated whether loss of NF1, but not RASA1, induced intrinsic EGF independence for CRC organoid survival by examining the effect of afatinib (EGFR/HERi) treatment on organoid viability by microscopy. Therefore, 5 days after trypsinization the parental P18T organoids, as well as the NF1 and RASA1 knock out organoid cultures, were filtered to homogenize their size and subsequently cultured with afatinib or DMSO (control) for 72 hours Figure . In agreFor both NF1 and RASA1 it has been demonstrated that they can affect RAS and ERK activity in various cell lines , 38, 45.Consistent with the phenotypes of our RASGAP knock out lines, only the loss of NF1 expression in CRC organoids resulted in enhanced ERK activation under basal conditions Figure , which wWhereas inhibition of EGF signaling clearly reduced the levels of active (phosphorylated) ERK in all three organoid types  after 1 hour of afatinib treatment, a substantial reactivation of ERK was observed in alive NF1 knock out organoids after 72 hours of afatinib treatment Figure  and 5B. Since the predicted function of NF1 GAP activity is to enhance the intrinsic GTP hydrolysis rate of GTP-bound RAS, we examined the amounts of active, GTP-bound RAS that are present in the absence of NF1. As a positive control, we also analyzed RAS-GTP levels in a P18T organoid line in which an activating mutation in KRAS (G12D) was introduced by CRISPR technology . In compRASA1 and NF1 knock out organoids. In addition to some clonal variability, we detected slight increased expression for most RASGAP genes in the case of NF1 or RASA1 deficiency  in comparison to wild type organoids, but not as evident as the resistance observed in KRAS mutant organoids Figure . WhereasWT and RASA1 knock out organoids remained small even upon drug withdrawal . Human CRC and TPO2  colon organoids were cultured as described previously . Culture medium containen advanced DMEM/F12 medium (Invitrogen) with 1% Penicillin/Streptomycin , 1% Hepes buffer (Invitrogen) and 1% Glutamax (Invitrogen), 20% R-spondin conditioned medium, 10% Noggin conditioned medium, 1x B27 (Invitrogen), 1.25 mM n-Acetyl Cysteine (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich), 50 ng/ml EGF (Invitrogen), 500 nM A83-01 (Tocris), 10 μM SB202190 (ApexBio) and 100 μg/ml Primorcin (Invitrogen). Organoids were splitted through Trypsin-EDTA (Sigma-Aldrich) treatment. Culture medium after splitting was supplemented with 10 μM Y-27632 dihydrochloride. For selection of RASGAP knock out mutants, organoids were grown in culture medium containing 1–2 μM puromycin or lacking EGF and containing 0.2–1.0 μM of afatinib .The patient-derived organoids derived in this study were previously established and characterized  and 1% Glutamax (Invitrogen).et al. (2015). Three days after transfection, culture media plus Y-27632 was exchanged with selection medium. After puromycin selection, surviving clones were picked and subjected to genotyping to detect the presence of insertions and deletions.The transfection protocol of P18T and TPO2 organoids was previously described in detail by Fujii For genotyping, genomic DNA was isolated using Viagen Direct PCR (Viagen). The presence of insertions or deletions in RASGAPs was verified by using the PCR product obtained using the following primers:NF1_fw 5′-GACCCTCTCCTTGCCTCTTC-3′, NF1_rv 5′-GGTGGCTCTGAAGCAGTTTC-3′, RASA1_fw1 5′-GACTCTTCCTTTTCCTCCCG-3′, RASA1_rv1 5′-G CAGTTTGGTGAGAGCCATG-3′, RASA1_fw2 5′-GT TGGGCATTACTGTGCTG-3′, RASA1_rv2 5′- GGTG GTGCAACTGGGTAAAG-3′, NF1_fw GAP domain 5′-GTACACTGTTAAATCTCAGG-3′, NF1_rv GAP domain 5′-AGAGGATGTGATCACAATTC-3′, RASA1_fw GAP domain 5′-GTCTTACAGAGTTAAGTCTG-3′,RASA1_rv GAP domain 5′-GTATTACAGACAGGTGT AAC-3′, RASA2_fw GAP domain 5′-CTTATGCCTTCT AGTATGTC-3′, RASA2_rv GAP domain 5′-TGCTTCTA AAGTGTTCAGTC-3′, RASA3_fw GAP domain 5′-GT GTTGACACAGGACGGTTC-3′, RASA3_rv GAP domain 5′-GGAGTACACAGGGAACATCC-3′,RASA4_fw GAP domain 5′-AGAACACTGGGAGGTG TTTG-3′, RASA4_rv GAP domain 5′-CGAACTCCTG ACCTTAAGTG-3′, RASAL1_fw GAP domain 5′-TGTG CACCTCCAGACAGTTG-3′, RASAL1_rv GAP domain 5′-GACCATGCAGGAAGAGGTTC-3′, RASAL2_fw GAP domain 5′-CAGCATTTCCAGGATG TCTG-3′, RASAL2_rv GAP domain 5′-AGCAGTGTA TGCTGACAAGG-3′, RASAL3_fw GAP domain 5′-GC CTAAGCATCAGCTACAAG-3′, RASAL3_rv GAP domain 5′-GTCTTCAGGTTATTCCGGAG-3′,SYNGAP1_fw GAP domain 5′-CACATCCTGCAGAGT ACAGG-3′, SYNGAP1_rv GAP domain 5′-ACAAGAG GGTGTGGTCACAC-3′, DAB2IP_fw GAP domain 5′-C ACCAGTTCTAGGCTCCTAC-3′, DAB2IP_rv GAP domain 5′-ACTTGCTGGGATCCACTTCG-3′.Products were sequenced using the following primers:NF1 exon 1 5′-CTTCCTTTCCTCCAGAGCCTG-3′,RASA1 exon 1 5′-CACAAGCTGCCCTCTCCCTT-3′,RASA1 exon 3 5′-CAAATAAACTTTGAGTGGTA-3′,NF1 GAP domain 5′-GTACACTGTTAAATCTCAGG-3′,RASA1 GAP domain 5′-GTACTTTCAACGCTGCAC-3′, RASA2 GAP domain 5′-CCTTCCCATCAATAGATC-3′,RASA3 GAP domain 5′-GTGTTGACACAGGACGGT TC-3′, RASA4 GAP domain 5′-GAACACTGGAGTCGA AGTC-3′, RASAL1 GAP domain 5′-CTGGAAGAATC ATGACTCC-3′, RASAL2 GAP domain 5′-CCAGTGCG TCATGAAGATAC-3′, RASAL3 GAP domain 5′-GTCT TCAGGTTATTCCGGAG-3′, SYNGAP1 GAP domain 5′CACGAGATTGGGTTGTGC-3′, DAB2IP GAP domain 5′-CTAGGTCTGGAATCCTAG-3′.In addition, the CloneJET PCR Cloning Kit was used to confirm indel generation in NF1 knock outs #1 and #6 and of RASA1 knock outs #1 and #3.The presence of the puromycin selection cassette was verified by using the PCR product obtained using primers:Puro_1_fw 5′-GACCCTCTCCTTGCCTCTTC-3′,Puro_1_rv 5′-GTTGGCGCCTACCGGTGG-3′,Puro_2_fw 5′-ATGGGGACCGAGTACAAGCC-3′,Puro_2_rv 5′-GTCGAAGATGAGGGTGAG-3′.http://crispr.mit.edu). The sgRNA guide sequences used can be found in the Supplementary Materials .The CRISPR guide RNA (sgRNAs) were designed by an online CRISPR design tool . For the generation of the donor template, genomic DNA from P18T organoids was used to PCR amplify the NF1 and RASA1 homology arms using high-fidelity Phusion Polymerase (New England BioLabs). The 5′ homology arm of RASA1 spans the region Chr5:87331757-87332526, and the 3′ homology arm spans the region Chr5:87332575-87333379. The 5′ homology arm of NF1 spans the region Chr17:31094574-31095323, and the 3′ homology arm spans the region Chr17:31095361-31096136. The homology arms were cloned into a pBlueScript plasmid expressing a 3229-bp AATPB:PGKpuroDtk selection cassette .For CRISPR-mediated homologous recombination the human codon-optimized Cas9 expression plasmid was obtained from Addgene (41815). The sgRNA-GFP plasmid was obtained from Addgene (41819) and used as a template for generating target specific sgRNAs as described in detail by Drost Prior to cell lysis, organoids were incubated with 1 mg/ml dispase II (Invitrogen) for 10 minutes at 37° C to digest the BME. Western blot samples for NF1 and RASA1 protein levels were lysed using NETN buffer  containing Complete protease inhibitors (Roche). Western blot samples for phosphorylated ERK were lysed using RIPA buffer  containing Complete protease inhibitors (Roche). Protein content was quantified using a BCA protein assay kit (Pierce™) and analyzed by Western blotting. Membranes were blocked and probed with antibodies directed against NF1 (RRID:AB_2149790), RASA1 (RRID:AB_303418), Vinculin RRID:AB_477629, a-tubulin (RRID:AB_477579), GAPDH (RRID:AB_2107445), pERK (RRID:AB_331646), and ERK (RRID:AB_390779).Samples for RAS-GTP isolation were lysed using Ral lysis buffer  containing Complete protease inhibitors (Roche). Lysates were normalized for protein levels using a BCA protein assay kit (Pierce™) and subsequently GTP-bound RAS was isolated via immunoprecipitation using recombinant RAS binding domain of RAF1 (RAF1-RBD). Protein lysates were run on SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked and probed with antibodies directed against RAS (RRID:AB_397425). Organoid treatments: afatinib  1 μM, 1 h and 24 h or DMSO.Organoids were harvested in RLT lysis buffer and RNA was isolated using the Qiagen RNeasy kit (Qiagen) according to the manufacturer's instructions. Extracted RNA was used as a template for cDNA production using iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's protocol. qRT–PCR was performed using FastStart Universal SYBR Green Master mix (Roche) according to the manufacturer's protocol. Results were calculated by using the relative standard curve method. Primer sequences:B2M_fw 5′-GAGGCTATCCAGCGTACTCCA-3′,B2M_rv 5′-CGGCAGGCATACTCATCTTTT-3′,NF1_fw 5′-GGATCCTACCAGGTTAGAACCATC-3′,NF1_rv 5′-AGCTTTATTCAGTAGGGAGTGGC-3′,RASA1_fw 5′-AATGCAGGATCAAGAACAAG-3′,RASA1_rv 5′-AAGGCATCCTTTGTTTTACG-3′,RASA2_fw 5′-GACTTGTGTAATCACAGTGG-3′,RASA2_rv 5′-TACCCTGAACCTCTGAATTG-3′,RASA3_fw 5′-AAGAGTGTTGAGCAGCCCAT-3′,RASA3_rv 5′TAGAGAGGCTGGTCCCCTTTG-3′,RASA4_fw 5′-CAGCCGGGACGACGTTATC-3′,RASA4_rv 5′-CCACCCGCTGAAACCCTTAG-3′,RASAL1_fw 5′-CGTGCTGGATGAGGACACTG-3′,RASAL1_rv 5′-TCCCTGCTCAGCGAGATCTT3′,RASAL2_fw 5′-CCCAACTCCATGGACACTGC-3′,RASAL2_rv 5′-GGATGGAAGCCGAAAGCTCG-3′,RASAL3_1_fw 5′-GGATCCAGATCGGATGCCTG-3′,RASAL3_1_rv 5′-TCCCTAGAGCCCAGAGCAC-3′,RASAL3_2_fw 5′-AACAGAACCGGAGACTGCTG-3′,RASAL3_2_rv 5′-GCTCCAACCTGGCCTTTTTC-3′,RASAL3_3_fw 5′-GCTCAAGAGGCTGAAAGAG-3′,RASAL3_3_rv 5′-CAGGTCCAGTTCAGAGAGTG-3′,DAB2IP_fw 5′-CATCATCAGCAGGTTGATGTCC-3′,DAB2IP_rv 5′-AGCGGGCTTTTGTTTCTAATGC-3′,SYNGAP1_fw 5′-ATGCAAAGCTTTAAGGAGTC-3′,SYNGAP1_rv 5′-GTTCCTGATGAAGTTGTTACC-3′Afatinib and Selumetinib, were purchased from Selleck Chemicals. These compounds were dissolved in dimethylsulfoxide  and stored as 10 mM aliquots.Five days after organoid trypsinization, 1 mg/ml dispase II (Invitrogen) was added to the medium of the organoids and these were incubated for 15 min at 37° C to digest the BME. Subsequently, organoids were mechanically dissociated by pipetting, filtrated using a 40 μm nylon cell strainer , resuspended in 75% BME/growth medium (40 organoids/μl) prior plating of two 10 μl drops on Nunc™ Lab-Tek™ II Chamber Slide™ Systems. After plating culture medium containing either 1 μM of afatinib, 1 μM of selumetinib, a combination of 1 μM of afatinib and 1 μM of selumetinib or DMSO was added. The labtek plates were mounted on an inverted confocal laser scanning microscope (Leica SP8X) and imaged using a 10X objective. For visualization of cell viability, organoids were incubated with 16.2 μΜ Hoechst 33342 (Life Technologies) and 1.5 μM DRAQ7™  for 30 min at 37° C prior imaging.For the GAP domain knock out CRISPR screen, organoids were imaged by an inverted routine microscope (Nikon Eclipse TS100) using a 4X or 10X objective. For calculating organoid count and size, organoids were incubated for 45 minutes with 500 ml culture medium containing 5 μM calcein-green (Invitrogen). For the quantification of the organoid size and count, FIJI analysis software was used.


Optic cup morphogenesis is an intricate process. Especially, the formation of the optic fissure is not well understood. Persisting optic fissures, termed coloboma, are frequent causes for congenital blindness. Even though the defective fusion of the fissure margins is the most acknowledged reason for coloboma, highly variable morphologies of coloboma phenotypes argue for a diverse set of underlying pathomechanisms. Here, we investigate optic fissure morphogenesis in zebrafish to identify potential morphogenetic defects resulting in coloboma. We show that the formation of the optic fissure depends on tissue flow movements, integrated into the bilateral distal epithelial flow forming the optic cup. On the temporal side, the distal flow translates into a ventral perpendicular flow, shaping the temporal fissure margin. On the nasal side, however, the distal flow is complemented by tissue derived from the optic stalk, shaping the nasal fissure margin. Notably, a distinct population of TGFβ-signalling positive cells is translocated from the optic stalk into both fissure margins. Furthermore, we show that induced BMP signalling as well as Wnt-signalling inhibition result in morphogenetic defects of the optic fissure. Our data also indicate that morphogenesis is crucial for a proper positioning of pre-specified dorsal–ventral optic cup domains. During optic cup morphogenesis, next to a bending of the neuroepithelium driven by basal constriction [Eye morphogenesis in vertebrates is a complex process during which initially optic vesicles evaginate from the eye field, located within the late prosencephalon –3. Subsetriction ,21, dynatriction ,22–25. Itriction . We hypotriction . We proptriction .Danio rerio). We further analysed how this is affected by BMP and Wnt signalling. We find that under normal conditions, the bilateral neuroretinal flow [Here, we addressed the morphogenesis of the optic fissure and the assembly of the optic fissure margins using zebrafish  embryos. Kaede is a photo-convertible fluorescent protein. Kaede expression was induced after a heat shock applied at 11 hpf for 15 min. Specific domains of the developing eye were photo-converted from green to red fluorescence (a). Following the photo-converted cells over time, a gastrulation-like rearrangement of lens-averted cells into the lens-facing domain can be appreciated . Note the marked flat cells , located in the lens-averted domain, which over time reach the lens-facing domain of the optic cup and elongate (figure 1c–f). The flow over the distal rims, which has been analysed previously [figure 1g–i).To address the tissue dynamics during optic cup formation, we employed rescence a. Followeviously , is nicei, arrow). In a transition zone, the stalk is connected to the lens-averted domain of the optic cup in a triangular manner . This connection can be found on the nasal side and the morphology of this transition zone is changed over time .Notably, the optic vesicle is connected to the optic stalk i, arrow.r manner j, arrow.ver time j,k.2.2.tg). In the lateral perspective, the prospective neuroretinal tissue, located within the lens-averted layer of the temporal ventral domain, is nicely visible . In the lateral aspect, these prospective neuroretinal cells are moved over the distal rim into the lens-facing domain of the optic cup .After this global analysis of tissue dynamics, we focused on the region of the forming optic fissure. At first, we addressed the temporal fissure margin. To visualize the cell dynamics, we made use of a transgenic line with ubiquitous nuclear and membrane labelling . This ventral rim corresponds to the temporal fissure margin. After optic cup morphogenesis is complete, the lens-averted layer is made up of flattened cells, the prospective RPE cells .Furthermore, it can be appreciated that in the ventral aspect, prospective neuroretinal cells move over a ventral rim via a perpendicular flow into the temporal lens-facing domain of the optic cup  experiment. Three-dimensional volume analysis over time allowed us to follow individual single cells, artificially labelled with coloured dots, from the lens-averted domain on their way into the optic cup . The distal rim  and the ventral rim can be appreciated in the max projection of the three-dimensional volume. Individual cells were followed over the distal and distal–ventral rim (magenta and blue) and over the ventral rim (red). This indicates that cells enter the optic cup via a ventral perpendicular flow, which is a direct continuation of the distal flow [figure 2e).To facilitate a better individual cell tracking, we reduced the density of labelled nuclei by injecting RNA coding for H2BGFP into one cell at the 4- to 8-cell staged embryo in the context of a ubiquitous membrane labelling, achieved by injections of RNA coding for lyntdTomato into the zygote. In addition, we increased the resolution in tal flow  on the t2.3.tg(hsp70:kaede) line again. We first photo-converted in the upper transition zone (b) to address the onset of stalk contribution. It can be appreciated nicely that the upper domain of the transition zone is moving into the fissure margin over time (b–f), changing the overall morphology of the transition zone from a triangular shape to a flat shape , what we termed ‘twist’ of the optic stalk. In order to visualize the further extent of the optic stalk contribution, we specifically labelled a domain within the optic stalk by photo-conversion of Kaede using embryos of the tg(hsp70:kaede) line. The photo-converted domain was then followed in a three-dimensional volume over time (figure 3g–k). It can be appreciated nicely that also this distinctly labelled domain is over time integrated from the optic stalk into the nasal optic fissure margin . These data are in line with previous observations [figure 3l,m, #; electronic supplementary material, movie S6). The findings are summed up in a diagram (figure 3n). To provide further quantitative data for the stalk contribution, we performed another set of experiments in which we photo-converted parts of the stalk domain early and analysed the contribution of this domain in the optic cup later . We found a stalk contribution ranging from 10 to 30%, being higher in the proximal regions of the optic cup and decreasing distally .We have now established that on the temporal side, the ventral and the distal flow are directly linked . On the ion zone b to addrver time b–f, chanrvations ,28. Nota2.4.in vivo TGFβ-signalling reporter in zebrafish [in vivo time-lapse imaging of embryos of the tg(SBE:GFPcaax), injected with RNA coding for lyntdTomato. We identified a domain in the forebrain and optic stalk, in which TGFβ signalling was activated . We then followed cells of this domain over time. We found that the active TGFβ-signalling domain was extending through the optic stalk . In distal fissure domains, the reporter signal is lost over time, due to either a continuous flow of these cells into other domains or due to a reduction in signalling activity over time .We recently found that TGFβ signalling is essential for optic fissure fusion . In thisebrafish . Here, w2.5.At this point, we had a framework for optic fissure formation in the context of optic cup morphogenesis at hand. Next, we wanted to address whether this morphogenetic process is hampered in conditions which are known to result in coloboma with a vast cleft, and if so, how. To this end, we made use of two models. On the one hand, we made use of an artificial expression of bmp4. On the other hand, we made use of an inhibition of porcupine, a factor which is crucial for the secretion of active Wnt ligands ,30.2.5.1.fsta), which was found expressed in the nasal and temporal domain of the forming optic cup [fsta also in the forming ventral aspect of the optic cup. Notably, we find it expressed in the transition zone from the stalk to the lens-averted domain (a–c). We then crossed fish from tg with fish from tg(SBE::GFPcaax), both described previously [in vivo time-lapse imaging (d). bmp4 inductions starting at 17 hpf resulted in overt morphogenetic defects of the optic cup (e–h). The optic stalk and the TGFβ-signalling active domain within were found enlarged (e–h), compared to controls (e–h) and the connection of the optic stalk to the optic cup was found broadened. A clear symmetry breaking connection of the stalk to a future nasal domain including a twist of the optic stalk could not be identified. Importantly, the optic stalk was found stuck and not integrated into the optic cup . Nevertheless, under these conditions without proper tissue dynamics, TGFβ-signalling active domains were later found within the optic cup but also within lens-averted domains (f–h). Notably, TGFβ-signalling activity within the optic cup can be observed during retinal differentiation (not shown). The perpendicular flow on the temporal domain was also largely absent . Notably, however, the distal flow in the ventral domains was not abrogated, explaining that in distal domains, an optic fissure was forming . This was at odds with findings from embryos in which bmp4 was driven from the rx2 cis-regulatory element, in which especially the distal flow on the temporal side was arrested [bmp4 at 13 hpf. Subsequently, the embryos were subjected to in vivo time-lapse imaging . Large ectopic putative neuroretinal domains can be appreciated . Importantly, also the stalk-derived flow as well as the perpendicular flow on the temporal side were found absent resulting in an absence of the optic fissure . Accordingly, the optic fissure is not only an important entry route for the POM and blood vessels, which enter through the fissure, but also for prospective neuroretinal tissue which is integrated into the ventral optic cup via the optic fissure margins. Thus, it is comprehensible that large domains of the ventral optic cup must be missing. We therefore addressed the ocular phenotype resulting from a bmp4 induction via heat shock 13 hpf at a later time point. Notably, at 40 hpf, the lens is protruding ventrally, explainable by missing ventral optic cup domains . So, even though no optic fissure was forming, at later developmental stages, a ventral gap is visible, not corresponding to the normal optic fissure. Since the overall flow movements were arrested drastically, the shape of the optic cup including the ‘abnormal ventral fissure’ must be resulting predominantly from a bending of the cup, probably caused by ojoplano-mediated basal constriction [First, we addressed the effect of induced BMP signalling. It was previously shown that the bilateral distal flow could be precociously arrested by induced BMP expression , the likptic cup . We now d domain a–c. We teviously , to prod imaging d. bmp4 iptic cup e–h. The enlarged e–h, compcontrols e–h and t domains f–h. Notaarrested   and also made use of an established Wnt reporter in zebrafish, tg(7xTCF-Xla.Siam:GFP) . A reduced Wnt-signalling activity can be appreciated nicely. Strikingly, and according to our hypothesis, the inhibition of porcupine was also resulting in a defect of the flow movements during optic cup morphogenesis . However, even more strikingly, the domain of TGFβ-signalling positive cells was found largely stuck within the optic stalk . In addition, this also negatively affected the ventral perpendicular flow on the temporal side . Moreover, we quantified distinct parameters (figure 6k). While the width of the optic cup was unchanged, its length was reduced. We also noted an increased width of the optic fissure, while its length was reduced. Overall, our data demonstrate that Wnt ligands are important for optic cup and optic fissure morphogenesis. The data furthermore indicate that the coloboma phenotype resulting from a hampered Wnt signalling is a secondary, morphogenetic coloboma.We next addressed the role of Wnt signalling during optic cup and fissure formation. In a mouse knockout, the loss of porcupine has been shown to result in a coloboma with a vast cleft and ectopic neuroretina . We inhi2.6.bmp4 expression and porcupine inhibition. However, next to affecting morphogenetic tissue dynamics, BMP and Wnt-signalling both could affect the dorsal ventral axis within the optic cup [bmp4-induced signalling and Wnt-signalling inhibition was also affecting the identity of cells within the optic cup. We addressed the expression of several markers, known to be expressed in distinct domains of the optic cup, namely bambi [vax2 [pax2a [bmp4 induction, performed at 17 hpf, resulted in a dramatic increase in the bambia expression domain. While bambia can be found expressed in the dorsal domain of the optic cup in controls (a–c), bmp4 induction increased the expression domain to the entire optic cup, the optic stalk and large domains of the forebrain (d–f). The expression of vax2, which in controls can be found in the ventral domain of the optic cup and optic stalk (g–i), was largely absent after bmp4 induction (j–l). Noteworthy, however, was a remaining expression of vax2 in the lens-averted domain on the temporal side (j). This domain is corresponding to the domain which was stuck during morphogenesis and was not correctly integrated into the lens-facing side of the optic cup (j). The induction of bmp4 furthermore resulted in a reduced pax2a expression domain within the optic cup . The pax2a expression domain in the optic stalk, however, was found enlarged . Notably, we found bambia expressed to a similar extent at 17 hpf in controls and porcupine-inhibited embryos (a–f), suggesting an onset of repression in between 17 hpf and 24 hpf. Vax2 was found expressed in the ventral optic cup and optic stalk also after porcupine inhibition (figure 8j–l), in domains where it can also be found in controls (figure 8g–i). Importantly, vax2 could also be found in lens-averted domains of the optic cup (figure 8j–l). These domains corresponded to the domains which failed to be integrated into the lens-facing domain of the optic cup correctly during morphogenesis. This is indicating that the domain of vax2, often used as a ventral marker for the optic cup, is largely originating from the lens-averted part of the early optic cup. We further analysed this by addressing the vax2 expression domain at 17 hpf (g–l). It can be appreciated nicely that in controls, but also in porcupine-inhibited embryos, the vax2-positive domains can be found in the lens-averted domain of the early optic cup (g–l). The domain of pax2a, expressed in the ventralmost optic cup and the optic stalk in controls, was found mildly reduced after inhibition of porcupine (figure 8m–r). Notably, after porcupine inhibition, pax2a expressing cells could be found in the lens-averted domain on the nasal side (figure 8p–r). An extension of the pax2a domain from the optic stalk into the nasal lens-averted domain resulting from porcupine inhibition could be seen already at 17 hpf (p–r). See figure 9m–o as control.The inhibition of porcupine resulted in a reduced expression intensity of  embryos a–f, suggt 17 hpf g–l. It cptic cup g–l. The t 17 hpf p–r. See 2.6.1.in vivo time-lapse analyses of zebrafish embryos that tissue dynamics play a major role for optic fissure formation .3.3.1.Husbandry. Zebrafish (Danio rerio) were kept in closed stocks in accordance with local animal welfare law. The fish facility is under supervision of the local representative of the animal welfare agency. Fish were maintained in a constant recirculating system at 28°C on a 12 L : 12 D cycle. Age of zebrafish embryos was determined according to Kimmel et al. [l et al. .: (tg(SBE:GFPcaax)Transgenic zebrafish. TGFβ reporter were generated previously, as described by Knickmeyer et al. [rx2 reporter: tg(Ola.rx2:eGFP-caax) were described and used previously [Ubiquitous nuclear and membrane reporter: The double transgenic line containing, tg(actb2:H2BGFP) tg(actb2:lyntdTomato) was created in the laboratory of Joachim Wittbrodt and kept in a AB/Casper background. Kaede reporter: tg was created in the laboratory of Jochen Wittbrodt and kept in a AB/WIK background. The two lines were kindly provided by the laboratory of Jochen Wittbrodt. Wnt reporter: The tg(7xTCF-Xla.Siam:GFP) transgenic line was kindly provided by Mathias Carl. HS-bmp4: tg were described as used previously [Rx2-driven bmp4 overexpression: tg was described as used previously [r et al. , rx2 repeviously . Ubiquiteviously . Rx2-drieviously .Transient labelling of zebrafish. Where indicated, H2BGFP  (50 ng µl−1), lyntdTomato (75–150 ng µl−1) was injected into 1- to 8-cell staged zebrafish embryos enabling four-dimensional imaging of mosaic or ubiquitous labelled zebrafish.3.2.tg and tg lines were applied in a heating block at 37°C for different times. Early heat shocks up to 13 hpf were applied for 15 min, later they were applied for 1 h.Heat shocks for the tg embryos, heat shocked wild-type siblings from the same clutch of eggs were used as controls.For 3.3.Porcupine inhibition: LGK-974 (Hycultec GmbH Beutelsbach) was solved in DMSO (10 mM). Embryos were treated with 20 µM LGK-974 from 13 hpf on until end of imaging. For the dose–response curve, embryos were treated with 1, 10, 20 and 50 µM of inhibitor. Control embryos were treated with an equal amount of DMSO without inhibitor.3.4.in situ hybridization (WMISH) was performed according to Quiring et al. [et al. [−1). For each in situ 8–10 embryos were stained, 4–6 were confocally imaged.Whole-mount g et al.  and Heer [et al. . WMISHs 3.5.in situs were imaged with a Leica TCS SP8 confocal microscope with a z-spacing of 3 µm.Whole-mount 3.6.z-spacing. The left and right eyes were used and oriented to fit the standard lateral view.Time-lapse imaging was performed on inverted Leica TCS SP8 set-ups with two internal hybrid detectors, and a 40× long distance objective (water immersion), using Immersol (Carl Zeiss) as immersion medium. For time-lapse imaging, embryos at appropriate stages were embedded in 1% low melting agarose in glass bottom dishes  and covered with zebrafish medium, including tricaine for anaesthesia. Confocal stacks were taken every 10 min with a resolution of 1024 × 1024 and 3 µm 3.7.The stacks were evaluated with FIJI software . For den3.8.Time-lapse SPIM imaging was performed with a Leica TCS SP8 set-up upgraded with a DLS . A 5× illumination objective, a 25× dipping lens together with 2.5 mm mirror caps were used to obtain a lightsheet. A stack was taken every 5 min. For imaging, glass-bottom-dishes (MatTek) were coated with 2% Agarose. Embryos were embedded in 1% low melting agarose and placed on the coating. Two notches were cut in the agarose with razorblades, so that the embryo was placed on a 2 mm wide stripe that could fit between the mirror caps. The embryo was covered with zebrafish medium, including tricaine for anaesthesia. Data from left and right eyes were used and oriented to fit the standard lateral view.3.9.rackJ [mageJ plugins as in [Tracking of cells was performed with MTrackJ . Tracks ns as in . The ove3.10.z-stacks with 3 µm z-spacing were taken. The length of the fissure was measured on an optical section below the lens, in a straight line from the embryonic retina to the surface ectoderm. The width of the OF and the eye was measured, at 50% length of the OF. The width of the neuroretina was measured in extension of the length of the OF. The length of the eye is the addition of the retina and the length of the OF. For each condition, seven eyes were measured. To determine significance, an unpaired t-test with Welch's correction was used, with p < 0.05.To analyse the length of the optic fissure, DAPI and anti-β-catenin-stained embryos were embedded upside down in low melting agarose. 3.11.Kaede in the optic stalk was photo-converted in a way that only the stalk and as little as possible from the optic vesicle was red. The embryo was then imaged until the flow movements were over. From every third optical section (section thickness 4 µm), the area of the optic cup was measured using the measure tool in Fiji. In the same sections, the red area was also measured and a percentage of red area in the optic cup after fissure development was calculated. This was done in three animals.