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Less, but more: Massive gene losses and expansions in appendicularians stretch the evolutionary limits of the FGF signalling pathway within chordates

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Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística

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[eng] The extensive diversity of forms in the animal kingdom arises from a limited set of evolutionarily conserved genes that coordinate during embryonic development to build complex bodies. Consequently, minor variations in this conserved set can lead to morphological innovations. The impact of gene loss on the evolution of developmental mechanisms has become an important topic in EvoDevo, especially since genomics revealed a high prevalence of gene loss across the tree of life. Over the past decade, the appendicularian Oikopleura dioica has emerged as a key model for studying this phenomenon in chordates. Despite a drastic reduction in its genome size and the loss of many developmental genes, O. dioica retains a typical chordate body plan, oﬀering a unique opportunity to investigate the organization of gene regulatory networks (GRNs) in a biological system heavily aﬀected by gene loss.
Previous research has shown that O. dioica has experienced signiﬁcant losses in developmental signalling pathways, including the complete dismantling of the retinoic acid (RA) signalling machinery and a major reduction in the number of Wnt families. The extensive interactions between these pathways and the Fibroblast Growth Factor (FGF) signalling pathway in other chordates raise an interest in investigating the evolution of FGF signalling in this species. This study characterizes the FGF signalling pathway in O. dioica, revealing an unprecedented remodelling compared to other chordates. The species has lost six of the eight Fgf subfamilies present in the last common chordate ancestor, but the remaining two subfamilies have expanded along with the FgfR gene.
Phylogenetic analyses, combined with comparative studies on gene structure and conserved protein motifs, provide robust evidence that the 10 Fgf genes identiﬁed in O. dioica belong exclusively to two subfamilies: Fgf9/16/20 and Fgf11/12/13/14. Analyses of gene structure, putative functional motifs, and developmental expression paherns indicate functional diversiﬁcation of these paralogs, as well as of the FgfR gene, following gene expansion. Additionally, examination of the three main intracellular transduction pathways associated with FgfR activation—MAPK, PLCγ/PKC, and PI3K/AKT—reveals that their main components are preserved and expressed throughout O. dioica development. However, structural rearrangements in FGF signal transduction have occurred, including the loss of classical Ras and Spred genes. Functional studies using the pharmacological inhibitor SU5402 demonstrate that the FGF signalling is crucial for embryonic development in O. dioica. Phenotypic analyses via whole-mount in situ hybridization of tissue-speciﬁc genes and omic approaches reveal that FGF signalling is involved in
 
gastrulation and cellular lineage diﬀerentiation. Finally, by comparing the developmental expression domains of O. dioica Fgf genes with those in the ascidian Ciona robusta, an evolutionary scenario is proposed where the evolution of FGF signalling in O. dioica is related to the transition from an ascidian-like biphasic lifestyle to a fully free-living one. This scenario categorizes expression domain changes into three types: extinction of ancestral domains due to gene loss, functional shuﬄing among surviving paralogs, and innovation of novel expression domains in new paralogs.
Overall, this doctoral thesis presents a comprehensive study of the FGF signalling pathway in a chordate with unprecedented levels of developmental gene loss. Our ﬁndings unveil the evolution of the Fgf family in appendicularians as a paradigmatic example of what we call “less, but more”, where massive gene losses, but also extensive duplications, result in the loss, conservation, and innovation of Fgf expression domains. This research provides new insights into the ﬂexibility and resilience of the FGF signalling pathway in chordates and raises questions about the evolutionary signiﬁcance of the Fgf9/16/20 and Fgf11/12/13/14 subfamilies. These ﬁndings contribute to a broader understanding of gene loss in EvoDevo, highlighting the complex interplay between genetic conservation and innovation.

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Tesis Doctorals - Departament - Genètica, Microbiologia i Estadística

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Less, but more: Massive gene losses and expansions in appendicularians stretch the evolutionary limits of the FGF signalling pathway within chordates