Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterizing the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera , a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, probably a result of non-homologous end joining, and a large deletion in the 3′ untranslated region due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.
Pattern formation in development has been principally studied in tissues that are not undergoing extensive cellular rearrangement. However, in most developmental contexts, gene expression domains emerge as cells re-arrange their spatial positions within the tissue, providing an additional, and seldom explored, level of complexity to the process of pattern formation in vivo. To investigate this issue, we addressed the regulation of TBox expression in the pre-somitic mesoderm (PSM) as this tissue develops in zebrafish embryos. Here, cells must differentiate in a manner that leads to well-defined spatial gene expression domains along the tissue while undergoing rapid movements to generate axial length. We find that in vivo, mesoderm progenitors undergo TBox differentiation over a broad range of time scales while in vitro their differentiation is simultaneous. By reverse-engineering a gene regulatory network (GRN)to recapitulate TBox gene expression, we were able to predict the population-level differentiation dynamics observed in culture, but not in vivo. In order to address this discrepancy in differentiation dynamics we developed a Live Modelling framework that allowed us to simulate the GRN on 3D tracking data generated from large-scale time-lapse imaging datasets of the develop-ing PSM. Once the network was simulated on a realistic representation of the cells morphogenetic context, the model was able to recapitulate the range of differentiation time scales observed in vivo, and revealed that these were necessary for TBox gene expression patterns to emerge correctly at the level of the tissue. This work thus highlights a previously unappreciated role for cell movement as a driver of pattern formation in development.
Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterising the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera, a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, likely a result of non-homologous end joining, and a large deletion in the 3′ UTR due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.
Vertebrate pigmentation patterns are highly diverse, yet we have a limited understanding of how evolutionary changes to genetic, cellular, and developmental mechanisms generate variation. To address this, we examine the formation of a sexually-selected male ornament exhibiting inter- and intra-specific variation, the egg-spot pattern, consisting of circular yellow-orange markings on the male anal fins of haplochromine cichlid fishes. We focus on Astatotilapia calliptera, the ancestor-type species of the Malawi cichlid adaptive radiation of over 850 species. We identify a key role for iridophores in initialising egg-spot aggregations composed of iridophore-xanthophore associations. Despite adult sexual dimorphism, aggregations initially form in both males and females, with development only diverging between the sexes at later stages. Unexpectedly, we found that the timing of egg-spot initialisation is plastic. The earlier individuals establish their own territory the earlier the aggregations form, with iridophores being the cell type that responds to social conditions. Furthermore, we observe apparent competitive interactions between adjacent egg-spot aggregations, which strongly suggests that egg-spot patterning results mostly from cell-autonomous cellular interactions. Together, these results demonstrate that A. calliptera egg-spot development is an exciting model for investigating pigment pattern formation at the cellular level in a system with developmental plasticity, sexual dimorphism, and intra-specific variation. As A. calliptera represents the ancestral bauplan for egg-spots, these findings provide a baseline for informed comparisons across the incredibly diverse Malawi cichlid radiation.
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