Genome editing in East African cichlids and tilapias: state-of-the-art and future directions.

African cichlid fishes of the Cichlidae family are a group of teleosts important for aquaculture and research. A thriving research community is particularly interested in the cichlid radiations of the East African Great Lakes. One key goal is to pinpoint genetic variation underlying phenotypic diversification, but the lack of genetic tools has precluded thorough dissection of the genetic basis of relevant traits in cichlids. Genome editing technologies are well established in teleost models like zebrafish and medaka. However, this is not the case for emerging model organisms, such as East African cichlids, where these technologies remain inaccessible to most laboratories, due in part to limited exchange of knowledge and expertise. The Cichlid Science 2022 meeting (Cambridge, UK) hosted for the first time a Genome Editing Workshop, where the community discussed recent advances in genome editing, with an emphasis on CRISPR/Cas9 technologies. Based on the workshop findings and discussions, in this review we define the state-of-the-art of cichlid genome editing, share resources and protocols, and propose new possible avenues to further expand the cichlid genome editing toolkit.

Mechanisms Underlying the Formation and Evolution of Vertebrate Color Patterns.

Vertebrates exhibit a wide range of color patterns, which play critical roles in mediating intra- and interspecific communication. Because of their diversity and visual accessibility, color patterns offer a unique and fascinating window into the processes underlying biological organization. In this review, we focus on describing many of the general principles governing the formation and evolution of color patterns in different vertebrate groups. We characterize the types of patterns, review the molecular and developmental mechanisms by which they originate, and discuss their role in constraining or facilitating evolutionary change. Lastly, we outline outstanding questions in the field and discuss different approaches that can be used to address them. Overall, we provide a unifying conceptual framework among vertebrate systems that may guide research into naturally evolved mechanisms underlying color pattern formation and evolution.

Toward a universal measure of robustness across model organs and systems.

The development of an individual must be capable of resisting the harmful effects of internal and external perturbations. This capacity, called robustness, can make the difference between normal variation and disease. Some systems and organs are more resilient in their capacity to correct the effects of internal disturbances such as mutations. Similarly, organs and organisms differ in their capacity to be resilient against external disturbances, such as changes in temperature. Furthermore, all developmental systems must be somewhat flexible to permit evolutionary change, and understanding robustness requires a comparative framework. Over the last decades, most research on developmental robustness has been focusing on specific model systems and organs. Hence, we lack tools that would allow cross-species and cross-organ comparisons. Here, we emphasize the need for a uniform framework to experimentally test and quantify robustness across study systems and suggest that the analysis of fluctuating asymmetry might be a powerful proxy to do so. Such a comparative framework will ultimately help to resolve why and how organs of the same and different species differ in their sensitivity to internal (e.g., mutations) and external (e.g., temperature) perturbations and at what level of biological organization buffering capacities exist and therefore create robustness of the developmental system.

East African cichlid fishes.

Cichlid fishes are a very diverse and species-rich family of teleost fishes that inhabit lakes and rivers of India, Africa, and South and Central America. Research has largely focused on East African cichlids of the Rift Lakes Tanganyika, Malawi, and Victoria that constitute the biodiversity hotspots of cichlid fishes. Here, we give an overview of the study system, research questions, and methodologies. Research on cichlid fishes spans many disciplines including ecology, evolution, physiology, genetics, development, and behavioral biology. In this review, we focus on a range of organismal traits, including coloration phenotypes, trophic adaptations, appendages like fins and scales, sensory systems, sex, brains, and behaviors. Moreover, we discuss studies on cichlid phylogenies, plasticity, and general evolutionary patterns, ranging from convergence to speciation rates and the proximate and ultimate mechanisms underlying these processes. From a methodological viewpoint, the last decade has brought great advances in cichlid fish research, particularly through the advent of affordable deep sequencing and advances in genetic manipulations. The ability to integrate across traits and research disciplines, ranging from developmental biology to ecology and evolution, makes cichlid fishes a fascinating research system.

Molecular parallelism in the evolution of a master sex-determining role for the anti-Mullerian hormone receptor 2 gene (amhr2) in Midas cichlids.

The evolution of sex chromosomes and their differentiation from autosomes is a major event during genome evolution that happened many times in several lineages. The repeated evolution and lability of sex-determination mechanisms in fishes makes this a well-suited system to test for general patterns in evolution. According to current theory, differentiation is triggered by the suppression of recombination following the evolution of a new master sex-determining gene. However, the molecular mechanisms that establish recombination suppression are known from few examples, owing to the intrinsic difficulties of assembling sex-determining regions (SDRs). The development of forward-genetics and long-read sequencing have generated a wealth of data questioning central aspects of the current theory. Here, we demonstrate that sex in Midas cichlids is determined by an XY system, and identify and assemble the SDR by combining forward-genetics, long-read sequencing and optical mapping. We show how long-reads aid in the detection of artefacts in genotype-phenotype mapping that arise from incomplete genome assemblies. The male-specific region is restricted to a 100-kb segment on chromosome 4 that harbours transposable elements and a Y-specific duplicate of the anti-Mullerian receptor 2 gene, which has evolved master sex-determining functions repeatedly. Our data suggest that amhr2Y originated by an interchromosomal translocation from chromosome 20 to 4 pre-dating the split of Midas and Flier cichlids. In the latter, it is pseudogenized and translocated to another chromosome. Duplication of anti-Mullerian genes is a common route to establishing new sex determiners, highlighting the role of molecular parallelism in the evolution of sex determination.

Comparative ontogenetic and transcriptomic analyses shed light on color pattern divergence in cichlid fishes.

Stripe patterns are a striking example for a repeatedly evolved color pattern. In the African adaptive radiations of cichlid fishes, stripes evolved several times independently. Previously, it has been suggested that regulatory evolution of a single gene, agouti-related-peptide 2 (agrp2), explains the evolutionary lability of this trait. Here, using a comparative transcriptomic approach, we performed comparisons between (adult) striped and nonstriped cichlid fishes of representatives of Lake Victoria and the two major clades of Lake Malawi (mbuna and non-mbuna lineage). We identify agrp2 to be differentially expressed across all pairwise comparisons, reaffirming its association with stripe pattern divergence. We therefore also provide evidence that agrp2 is associated with the loss of the nonstereotypic oblique stripe of Mylochromis mola. Complementary ontogenetic data give insights into the development of stripe patterns as well as vertical bar patterns that both develop postembryonically. Lastly, using the Lake Victoria species pair Haplochromis sauvagei and Pundamilia nyererei, we investigated the differences between melanic and non-melanic regions to identify additional genes that contribute to the formation of stripes. Expression differences-that most importantly also do not include agrp2-are surprisingly small. This suggests, at least in this species pair, that the stripe phenotype might be caused by a combination of more subtle transcriptomic differences or cellular changes without transcriptional correlates. In summary, our comprehensive analysis highlights the ontogenetic and adult transcriptomic differences between cichlids with different color patterns and serves as a basis for further investigation of the mechanistic underpinnings of their diversification.

Benefits and limitations of a new genome-based PCR-RFLP genotyping assay (GB-RFLP): A SNP-based detection method for identification of species in extremely young adaptive radiations.

High-throughput DNA sequencing technologies make it possible now to sequence entire genomes relatively easily. Complete genomic information obtained by whole-genome resequencing (WGS) can aid in identifying and delineating species even if they are extremely young, cryptic, or morphologically difficult to discern and closely related. Yet, for taxonomic or conservation biology purposes, WGS can remain cost-prohibitive, too time-consuming, and often constitute a "data overkill." Rapid and reliable identification of species (and populations) that is also cost-effective is made possible by species-specific markers that can be discovered by WGS. Based on WGS data, we designed a PCR restriction fragment length polymorphism (PCR-RFLP) assay for 19 Neotropical Midas cichlid populations (Amphilophus cf. citrinellus), that includes all 13 described species of this species complex. Our work illustrates that identification of species and populations (i.e., fish from different lakes) can be greatly improved by designing genetic markers using available "high resolution" genomic information. Yet, our work also shows that even in the best-case scenario, when whole-genome resequencing information is available, unequivocal assignments remain challenging when species or populations diverged very recently, or gene flow persists. In summary, we provide a comprehensive workflow on how to design RFPL markers based on genome resequencing data, how to test and evaluate their reliability, and discuss the benefits and pitfalls of our approach.

The repeated evolution of stripe patterns is correlated with body morphology in the adaptive radiations of East African cichlid fishes.

Color patterns are often linked to the behavioral and morphological characteristics of an animal, contributing to the effectiveness of such patterns as antipredatory strategies. Species-rich adaptive radiations, such as the freshwater fish family Cichlidae, provide an exciting opportunity to study trait correlations at a macroevolutionary scale. Cichlids are also well known for their diversity and repeated evolution of color patterns and body morphology. To study the evolutionary dynamics between color patterns and body morphology, we used an extensive dataset of 461 species. A phylogenetic supertree of these species shows that stripe patterns evolved ~70 times independently and were lost again ~30 times. Moreover, stripe patterns show strong signs of correlated evolution with body elongation, suggesting that the stripes' effectiveness as antipredatory strategy might differ depending on the body shape. Using pedigree-based analyses, we show that stripes and body elongation segregate independently, indicating that the two traits are not genetically linked. Their correlation in nature is therefore likely maintained by correlational selection. Lastly, by performing a mate preference assay using a striped CRISPR-Cas9 mutant of a nonstriped species, we show that females do not differentiate between striped CRISPR mutant males and nonstriped wild-type males, suggesting that these patterns might be less important for species recognition and mate choice. In summary, our study suggests that the massive rates of repeated evolution of stripe patterns are shaped by correlational selection with body elongation, but not by sexual selection.

An intronic transposon insertion associates with a trans-species color polymorphism in Midas cichlid fishes.

Polymorphisms have fascinated biologists for a long time, but their genetic underpinnings often remain elusive. Here, we aim to uncover the genetic basis of the gold/dark polymorphism that is eponymous of Midas cichlid fish (Amphilophus spp.) adaptive radiations in Nicaraguan crater lakes. While most Midas cichlids are of the melanic "dark morph", about 10% of individuals lose their melanic pigmentation during their ontogeny and transition into a conspicuous "gold morph". Using a new haplotype-resolved long-read assembly we discover an 8.2 kb, transposon-derived inverted repeat in an intron of an undescribed gene, which we term goldentouch in reference to the Greek myth of King Midas. The gene goldentouch is differentially expressed between morphs, presumably due to structural implications of inverted repeats in both DNA and/or RNA (cruciform and hairpin formation). The near-perfect association of the insertion with the phenotype across independent populations suggests that it likely underlies this trans-specific, stable polymorphism.

Of bars and stripes: A Malawi cichlid hybrid cross provides insights into genetic modularity and evolution of modifier loci underlying colour pattern diversification.

Understanding the origins of phenotypic diversity among closely related species remains an important largely unsolved question in evolutionary biology. With over 800 species, Lake Malawi haplochromine cichlid fishes are a prominent example of extremely fast evolution of diversity including variation in colouration. Previously, a single major effect gene, agrp2 (asip2b), has been linked to evolutionary losses and gains of horizontal stripe patterns in cichlids, but it remains unknown what causes more fine-scale variation in the number and continuity of the stripes. Also, the genetic basis of the most common colour pattern in African cichlids, vertical bars, and potential interactions between the two colour patterns remain unknown. Based on a hybrid cross of the horizontally striped Lake Malawi cichlid Pseudotropheus cyaneorhabdos and the vertically barred species Chindongo demasoni we investigated the genetic basis of both colour patterns. The distribution of phenotypes in the F2  generation of the cross indicates that horizontal stripes and vertical bars are independently inherited patterns that are caused by two sets of genetic modules. While horizontal stripes are largely controlled by few major effect loci, vertical bars are a highly polygenic trait. Horizontal stripes show substantial variation in the F2  generation that, interestingly, resemble naturally occurring phenotypes found in other Lake Malawi cichlid species. Quantitative trait loci (QTL) mapping of this cross reveals known (agrp2) and unknown loci underlying horizontal stripe patterns. These findings provide novel insights into the incremental fine-tuning of an adaptive trait that diversified through the evolution of additional modifier loci.

Functional conservation and divergence of color-pattern-related agouti family genes in teleost fishes.

While color patterns are highly diverse across the animal kingdom, certain patterns such as countershading and stripe patterns have evolved repeatedly. Across vertebrates, agouti-signaling genes have been associated with the evolution of both patterns. Here we study the functional conservation and divergence by investigating the expression patterns of the two color-pattern-related agouti-signaling genes, agouti-signaling protein 1 (asip1) and agouti-signaling protein 2b (asip2b, also known as agrp2) in Teleostei. We show that the dorsoventral expression profile of asip1 and the role of the "stripe repressor" asip2b are shared across multiple teleost lineages and uncover a previously unknown association between stripe-interstripe patterning and both asip1 and asip2b expression. In some species, including the zebrafish (Danio rerio), these two genes show complementary and overlapping expression patterns in line with functional redundancy. Our results thus suggest how conserved and novel functions of agouti-signaling genes might have shaped the evolution of color patterns across teleost fishes.

Different Sources of Allelic Variation Drove Repeated Color Pattern Divergence in Cichlid Fishes.

The adaptive radiations of East African cichlid fish in the Great Lakes Victoria, Malawi, and Tanganyika are well known for their diversity and repeatedly evolved phenotypes. Convergent evolution of melanic horizontal stripes has been linked to a single locus harboring the gene agouti-related peptide 2 (agrp2). However, where and when the causal variants underlying this trait evolved and how they drove phenotypic divergence remained unknown. To test the alternative hypotheses of standing genetic variation versus de novo mutations (independently originating in each radiation), we searched for shared signals of genomic divergence at the agrp2 locus. Although we discovered similar signatures of differentiation at the locus level, the haplotypes associated with stripe patterns are surprisingly different. In Lake Malawi, the highest associated alleles are located within and close to the 5' untranslated region of agrp2 and likely evolved through recent de novo mutations. In the younger Lake Victoria radiation, stripes are associated with two intronic regions overlapping with a previously reported cis-regulatory interval. The origin of these segregating haplotypes predates the Lake Victoria radiation because they are also found in more basal riverine and Lake Kivu species. This suggests that both segregating haplotypes were present as standing genetic variation at the onset of the Lake Victoria adaptive radiation with its more than 500 species and drove phenotypic divergence within the species flock. Therefore, both new (Lake Malawi) and ancient (Lake Victoria) allelic variation at the same locus fueled rapid and convergent phenotypic evolution.

Contrasting signatures of genomic divergence during sympatric speciation.

The transition from 'well-marked varieties' of a single species into 'well-defined species'-especially in the absence of geographic barriers to gene flow (sympatric speciation)-has puzzled evolutionary biologists ever since Darwin1,2. Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process3. Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs4, but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories5. Here, within a young species complex of neotropical cichlid fishes (Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry.

Neural innervation as a potential trigger of morphological color change and sexual dimorphism in cichlid fish.

Many species change their coloration during ontogeny or even as adults. Color change hereby often serves as sexual or status signal. The cellular and subcellular changes that drive color change and how they are orchestrated have been barely understood, but a deeper knowledge of the underlying processes is important to our understanding of how such plastic changes develop and evolve. Here we studied the color change of the Malawi golden cichlid (Melanchromis auratus). Females and subordinate males of this species are yellow and white with two prominent black stripes (yellow morph; female and non-breeding male coloration), while dominant males change their color and completely invert this pattern with the yellow and white regions becoming black, and the black stripes becoming white to iridescent blue (dark morph; male breeding coloration). A comparison of the two morphs reveals that substantial changes across multiple levels of biological organization underlie this polyphenism. These include changes in pigment cell (chromatophore) number, intracellular dispersal of pigments, and tilting of reflective platelets (iridosomes) within iridophores. At the transcriptional level, we find differences in pigmentation gene expression between these two color morphs but, surprisingly, 80% of the genes overexpressed in the dark morph relate to neuronal processes including synapse formation. Nerve fiber staining confirms that scales of the dark morph are indeed innervated by 1.3 to 2 times more axonal fibers. Our results might suggest an instructive role of nervous innervation orchestrating the complex cellular and ultrastructural changes that drive the morphological color change of this cichlid species.

Postmitotic Hoxa5 Expression Specifies Pontine Neuron Positional Identity and Input Connectivity of Cortical Afferent Subsets.

The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and sub-circuit assembly of PN neuron subsets in the cortico-cerebellar pathway.

Developmental and Cellular Basis of Vertical Bar Color Patterns in the East African Cichlid Fish Haplochromis latifasciatus.

The East African adaptive radiations of cichlid fishes are renowned for their diversity in coloration. Yet, the developmental basis of pigment pattern formation remains largely unknown. One of the most common melanic patterns in cichlid fishes are vertical bar patterns. Here we describe the ontogeny of this conspicuous pattern in the Lake Kyoga species Haplochromis latifasciatus. Beginning with the larval stages we tracked the formation of this stereotypic color pattern and discovered that its macroscopic appearance is largely explained by an increase in melanophore density and accumulation of melanin during the first 3 weeks post-fertilization. The embryonal analysis is complemented with cytological quantifications of pigment cells in adult scales and the dermis beneath the scales. In adults, melanic bars are characterized by a two to threefold higher density of melanophores than in the intervening yellow interbars. We found no strong support for differences in other pigment cell types such as xanthophores. Quantitative PCRs for twelve known pigmentation genes showed that expression of melanin synthesis genes tyr and tyrp1a is increased five to sixfold in melanic bars, while xanthophore and iridophore marker genes are not differentially expressed. In summary, we provide novel insights on how vertical bars, one of the most widespread vertebrate color patterns, are formed through dynamic control of melanophore density, melanin synthesis and melanosome dispersal.

The Cre/Lox System to Assess the Development of the Mouse Brain.

Cre-mediated recombination has become a powerful tool to confine gene deletions (conditional knockouts) or overexpression of genes (conditional knockin/overexpression). By spatiotemporal restriction of genetic manipulations, major problems of classical knockouts such as embryonic lethality or pleiotropy can be circumvented. Furthermore, Cre-mediated recombination has broad applications in the analysis of the cellular behavior of subpopulations and cell types as well as for genetic fate mapping. This chapter gives an overview about applications for the Cre/LoxP system and their execution.

Evolutionary Dynamics of Structural Variation at a Key Locus for Color Pattern Diversification in Cichlid Fishes.

Color patterns in African cichlid fishes vary spectacularly. Although phylogenetic analysis showed already 30 years ago that many color patterns evolved repeatedly in these adaptive radiations, only recently have we begun to understand the genomic basis of color variation. Horizontal stripe patterns evolved and were lost several times independently across the adaptive radiations of Lake Victoria, Malawi, and Tanganyika and regulatory evolution of agouti-related peptide 2 (agrp2/asip2b) has been linked to this phenotypically labile trait. Here, we asked whether the agrp2 locus exhibits particular characteristics that facilitate divergence in color patterns. Based on comparative genomic analyses, we discovered several recent duplications, insertions, and deletions. Interestingly, one of these events resulted in a tandem duplication of the last exon of agrp2. The duplication likely precedes the East African radiations that started 8-12 Ma, is not fixed within any of the radiations, and is found to vary even within some species. Moreover, we also observed variation in copy number (two to five copies) and secondary loss of the duplication, illustrating a surprising dynamic at this locus that possibly promoted functional divergence of agrp2. Our work suggests that such instances of exon duplications are a neglected mechanism potentially involved in the repeated evolution and diversification that deserves more attention.

Molecular mechanisms of convergent color pattern evolution.

Color patterns are a fascinating organismal feature and play key roles in several fundamental ecological and evolutionary processes. They give important insights into the process of phenotypic divergence. Color patterns also provide striking examples of convergence, the independent evolution of phenotypic similarities. In the era of genome sequencing and editing it is now increasingly possible to study divergence and convergence from a molecular perspective. Recent work in cichlid fishes suggests independent genetic changes at the same genomic locus as the cause for the parallel losses and gains of stripe color patterns. Together with evidence from other animal groups, a picture emerges whereby independent mutations at genomic hotspots associate with both the divergent and convergent evolution of genetically and physiologically distinct color pattern phenotypes such as stripe patterns and countershading.

Genome of the Malawi golden cichlid fish (Melanochromis auratus) reveals exon loss of oca2 in an amelanistic morph.

The tropical freshwater fish family Cichlidae is famous for its record-breaking rates of speciation and diversity in colors and color patterns. Here, we sequenced the genome of the Lake Malawi cichlid Melanochromis auratus to study the genetic basis of an amelanistic morph of this species that lacks the typical melanic stripes and markings. Genome sequencing of the amelanistic and wild-type morph revealed the loss of the second exon of the known pigmentation gene oculocutaneous albinism II (oca2), also known as p(ink-eyed dilution) gene or melanocyte-specific transporter gene. Additional genotyping confirms the complete association with this recessive Mendelian phenotype. The deletion results in a shorter transcript, lacking an acidic di-leucine domain that is crucial for trafficking of the Oca2 protein to melanosomes. The fact that oca2 is involved in a wide range of amelanistic morphs across vertebrates demonstrates its highly conserved function.