Analysis of Fox genes inSchmidtea mediterraneareveals new families and a conserved role ofSmed-foxOin controlling cell death

Abstract

The forkhead box (Fox) genes encode transcription factors that control several key aspects of development. Present in the ancestor of all eukaryotes, Fox genes underwent several duplications followed by loss and diversification events that gave rise to the current 25 families. However, few Fox members have been identified from the Lophotrochozoa clade, and specifically from planarians, which are a unique model for understanding development, due to the striking plasticity of the adult. The aim of this study was to identify and perform evolutionary and functional studies of the Fox genes of lophotrochozoan species and, specifically, of the planarian Schmidtea mediterranea. Generating a pipeline for identifying Forkhead domains and using phylogenetics allowed us the phylogenetic reconstruction of Fox genes. We corrected the annotation for misannotated genes and uncover a new family, the QD, present in all metazoans. According to the new phylogeny, the 27 Fox genes found in Schmidtea mediterranea were classified into 12 families. In Platyhelminthes, family losses were accompanied by extensive gene diversification and the appearance of specific families, the A(P) and N(P). Among the newly identified planarian Fox genes, we found a single copy of foxO, which shows an evolutionary conserved role in controlling cell death.Author summaryTranscription factors are the key elements that regulate gene expression in the nucleus. The forkhead box (Fox) transcription factors are one of the most numerous and they control key aspects of development. Fox genes were already present in the ancestor of all eukaryotes, and then underwent several duplications followed by loss and diversification events that gave rise to the current Fox families in the different species. The available data classifies Fox genes in 25 families, but they include few members corresponding to Lophotrocozoa, one of the two invertebrate phyla that includes annelids, molluscs or platyhelmintes. In this study we identify and perform evolutionary studies of the Fox genes of several lophotrochozoan species and, specifically, of the planarian Schmidtea mediterranea. The result is the correction of the annotation of Fox genes from many species, proposing a new nomenclature, and the identification of new families; the QD family, present in all metazoans, and the A(P) and N(P) families, specific of Platyhelminthes. We also study the function of Schmidtea mediterranea foxO, a gene involved in aging and cancer in other species, showing its evolutionary conserved role in controlling cell death according to cell metabolism.

Gene activation of metazoan Fox transcription factors at the onset of metamorphosis in the marine demosponge Amphimedon queenslandica

“…However, we note that some previously identified Fox subfamilies are not well supported in our analysis. This is similar to previous studies that have generated phylogenetic trees with varying topologies (e.g., Kaestner et al, 2000; Kaufmann & Knöchel, 1996; Larroux et al, 2008; Pascual‐Carreras et al, 2021; Schomburg et al, 2022; Shimeld, Boyle, et al, 2010a). For instance, our phylogeny supports FoxN1 / 4 and FoxN2 / 3 diverging from an ancestral holozoan FoxN gene, in contrast to previous analyses that support FoxN1 / 4 being an ancestral premetazoan gene and FoxN2 / 3 evolving after metazoans diverged from choanoflagellates (Larroux et al, 2008; Shimeld, Degnan, & Luke, 2010b).…”

“…Phylogenetic analysis of Forkhead domain sequences from 13 holozoans (Table S1) – one filasterean, two choanoflagellates, six poriferans, one ctenophore, one cnidarian, one annelid, and one chordate – is consistent with previous classifications of this family into two clades and 25–26 subfamilies (Figure 1) (Kaestner et al, 2000; Kaufmann & Knöchel, 1996; Larroux et al, 2008; Pascual‐Carreras et al, 2021; Schomburg et al, 2022; Shimeld, Boyle, et al, 2010a; Shimeld, Degnan, & Luke, 2010b). Clade I Fox genes are nested within Clade II.…”

“…As is the case with other metazoan transcription factor families, the specific functions of Fox genes have been elucidated largely from studies in Drosophila and vertebrate models (reviewed in Carlsson & Mahlapuu, 2002; Golson & Kaestner, 2016). Nonetheless, spatial and temporal expression in a range of eumetazoans are consistent with Fox genes regulating a wide range of developmental – including embryonic development, specification, and maintenance of cell state, cell proliferation, and differentiation – and metabolic processes (Carlsson & Mahlapuu, 2002; Fritzenwanker et al, 2014; Golson & Kaestner, 2016; Hannenhalli & Kaestner, 2009; Leclère et al, 2019; Magie et al, 2005; Pascual‐Carreras et al, 2021; Seudre et al, 2022; Tu et al, 2006). In some cases, specific Fox gene subfamilies appear to have a widely conserved function, such as FoxO that appears to regulate life spans from yeast to mammals (Finlay et al, 2019; Greer et al, 2007; Jiang et al, 2019).…”

“…The 408 genes co‐expressed with AmqFoxOa and AmqFoxN2 / 3 are also predominantly involved in metabolic processes and morphogenesis, consistent with the rapid body plan reorganization occurring at this time. FoxO in particular is associated with these cellular processes and with the regulation of stress and longevity and metabolism in other animals, which can be influenced by changes in extracellular signals (Carlsson & Mahlapuu, 2002; Golson & Kaestner, 2016; Kwak et al, 2018; Pascual‐Carreras et al, 2021; Tan et al, 1998; Yao et al, 2001). For example, FoxO3, FoxG, and FoxA cooperate to regulate cellular processes and stress responses in the sea urchin Paracentrotus lividus (Ruocco et al, 2017).…”

“…The number of Fox genes in the genomes of eumetazoans (cnidarians, insects, vertebrates, and other bilaterians) ranges from just under 20 to over 40. The eumetazoan Fox genes have been classified into 25–26 subfamilies that form two clades (Kaestner et al, 2000; Kaufmann & Knöchel, 1996; Larroux et al, 2008; Pascual‐Carreras et al, 2021; Schomburg et al, 2022; Shimeld, Boyle, et al, 2010a; Shimeld, Degnan, & Luke, 2010b). The more ancestral clade (Clade II) is comprised of both (1) subfamilies shared with non‐metazoans ( FoxJ1 , FoxJ2 / 3 , FoxM , FoxN1 / 4 ) and (2) metazoan‐specific subfamilies ( FoxN2 / 3 , FoxO , FoxP , FoxK ).…”