https://orcid.org/0000-0002-0027-2175
Figures
Abstract
Fox genes represent an evolutionary old class of transcription factor encoding genes that evolved in the last common ancestor of fungi and animals. They represent key-components of multiple gene regulatory networks (GRNs) that are essential for embryonic development. Most of our knowledge about the function of Fox genes comes from vertebrate research, and for arthropods the only comprehensive gene expression analysis is that of the fly Drosophila melanogaster. For other arthropods, only selected Fox genes have been investigated. In this study, we provide the first comprehensive gene expression analysis of arthropod Fox genes including representative species of all main groups of arthropods, Pancrustacea, Myriapoda and Chelicerata. We also provide the first comprehensive analysis of Fox gene expression in an onychophoran species. Our data show that many of the Fox genes likely retained their function during panarthropod evolution highlighting their importance in development. Comparison with published data from other groups of animals shows that this high degree of evolutionary conservation often dates back beyond the last common ancestor of Panarthropoda.
Citation: Janssen R, Schomburg C, Prpic N-M, Budd GE (2022) A comprehensive study of arthropod and onychophoran Fox gene expression patterns. PLoS ONE 17(7): e0270790. https://doi.org/10.1371/journal.pone.0270790
Editor: Michael Schubert, Laboratoire de Biologie du Développement de Villefranche-sur-Mer, FRANCE
Received: February 18, 2022; Accepted: June 20, 2022; Published: July 8, 2022
Copyright: © 2022 Janssen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Financial funding to GEB and RJ was provided by the Swedish Natural Science Council (VR), grant no. 621-2011-4703. Financial funding to NMP and CS was provided by the Deutsche Forschungsgemeinschaft (DFG; grant numbers PR1109/4-1, PR1109/6-1 and PR1109/7-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Fox gene transcription factors are characterized by the presence of an approximately 100 amino acid long DNA-binding motif, the so-called forkhead domain [1]. This domain forms three α-helices, three β-sheets and two wing-shaped structures. Fox genes are involved in various developmental processes and have been studied in a large number of animals including vertebrates [2–4], cephalochordates [5, 6], hemichordates [7], echinoderms [8], annelids [9], molluscs [9], cnidarians [10, 11] and even sponges [12, 13]. Among the Ecdysozoa, however, comprehensive studies are restricted to the dipteran fly Drosophila melanogaster [14, 15] and the nematode worm Caenorhabditis elegans [16] (and references therein) (see Table 1 for Drosophila gene names). Data from other groups of ecdysozoans and other arthropods are relatively sparse and often only address single Fox genes [e.g. 17–22].
The first unified nomenclature for Fox genes was established by [23], defining 15 classes of Fox genes. In the following years, additional classes have been identified and four classes, FoxJ, FoxL, FoxN, and FoxQ each were subdivided into two (e.g. FoxJ into FoxJ1 and FoxJ2) [24]. Two of these, FoxR and FoxS are believed to represent vertebrate specific groups [25, 26]. Recently, yet another class of Fox genes, FoxT, has been identified that appears to be panarthropod specific [27, 28].
In this study, we analyzed the embryonic expression patterns of Fox genes in three arthropod species, representing main branches of Arthropoda, the red flour beetle Tribolium castaneum, the pill millipede Glomeris marginata, the common house spider Parasteatoda tepidariorum, and as a representative of Onychophora, the blue velvet worm Euperipatoides kanangrensis. Together, these species cover most of Panarthropoda. Tribolium serves as a representative of Hexapoda, that in contrast to Drosophila shows a more ancestral mode of development (e.g. [29, 30]). Glomeris, as a representative of Myriapoda, represents the sister group to hexapods + crustaceans (Pancrustacea) with which they form the Mandibulata (Myriapoda + Pancrustacea). Parasteatoda (as a representative of Chelicerata) represents the sister group to Mandibulata, and Euperipatoides (as a representative of Onychophora) likely represents the closest related outgroup to Arthropoda (e.g. [31, 32]).
We analyzed the embryonic expression patterns of all identified Fox genes in these species (Fig 1). Whenever appropriate we also provide additional expression data on previously investigated Fox gene expression patterns. Expression data that simply add to or verify comprehensive earlier studies are provided in the supplementary data. In cases where a given Fox gene expression pattern has previously been investigated exhaustively, we refer to the published literature. Additionally, we compare the currently available data on Fox gene expression and function and try to recapitulate their potential roles during panarthropod evolution.
Modified after [28] (their Fig 2). Green and light green boxes indicate that the gene falls in a given class of Fox genes in all or two of three analyses conducted in [28], respectively. Roman numbers in boxes indicate numbers of paralogs. A dash (-) indicates a predicted loss of a given lineage-specific Fox class gene. Solid vertical grey bars suggest losses of a class of Fox genes. Data on Apis mellifera stem partially from [198]. Note that the lack of FoxJ23 in holometabolous insects in this overview does not represent a general loss in this group of animals (as it is present and some species), and that a possible FoxQ1 gene was identified in a scorpion, albeit with weaker support [28]. A recent report by [62] listed 21 Tribolium Fox genes, but note that this is because three and two identical sequences of FoxO and FoxP respectively were incorporated in their list [62] (their Fig 2). The same analysis lists two Drosophila FoxP genes which appears to be a typo in their figure.
Methods
Animal husbandry and embryo preparation
Embryos were treated as described in [33] (Tribolium), [34] (Glomeris), [35] (Parasteatoda), and [36] (Euperipatoides). Developmental stages are defined as per [37] (Tribolium), [34] (Glomeris), [38] (Parasteatoda), and [39] (Euperipatoides).
Sequence analysis
The phylogenetic relationship of all Fox genes identified in our research organisms has recently been investigated in [28]. For an overview of the Fox gene complements of arthropods and onychophorans, see Fig 1 (and S1 Fig).
Gene cloning
For all species, RNA isolation of a mix of embryos representing different developmental stages, and subsequent cDNA synthesis were carried out as described in [34]. All gene fragments were amplified using gene specific primers (S1 Table) based on published genomes and transcriptomes, and Topo-TA cloned into the pCRII vector (Invitrogen, Carlsbad, CA, USA). Sequences were checked on an ABI3730XL analyser using Big Dye dye-terminators by a commercial sequencing service (Macrogen, Korea). Sequences identifiers of all investigated panarthropod Fox genes are listed in S2 Table.
Whole-mount in-situ hybridization and DNA staining
All whole-mount in-situ hybridizations were performed as described in [40]. Cell nuclei were detected using 4-6-Diamidin-2-phenylindol (DAPI). Incubation in 2 μg/ml DAPI in phosphate buffered saline with 0.1% Tween-20 (PBST) for 30 minutes was followed by extensive washes in PBST to remove excess DAPI.
Data documentation
Embryos were photographed using a Leica DC490 digital camera equipped with a UV light source mounted onto a MZ-FLIII Leica dissection microscope. Brightness, contrast, and color values were adjusted in all images using the image processing software Adobe Photoshop CC 2018 (for Apple Macintosh (Adobe Systems Inc. San Jose, CA, USA).
Results
Gene expression patterns
FoxA.
Tribolium FoxA is first expressed in the yolk (not shown), and at later stages in the primordia of the stomodaeum and the proctodaeum (S2A Fig). Additional expression appears along the ventral midline in segmental clusters (S2A–S2C Fig, slim arrow) and laterally in the head lobes (S2A–S2C Fig, short arrow). At later developmental stages, after germ band retraction, it is expressed in the brain and in the hindgut, including the Malpighian tubules (S2 Fig). Some aspects of FoxA expression in Tribolium have been reported previously by [41].
Glomeris FoxA is first expressed in the primordia of the hindgut and the foregut. When the proctodaeum and the stomodaeum form, FoxA expressing cells sink in and form the through gut (S3A–S3D Fig). At late developmental stages, additional expression appears in the ventral nervous system (VNS) (S3D Fig, arrow). For further descriptions of Glomeris FoxA expression, see also [42].
First expression of Parasteatoda FoxA-1 (described as FoxA in [43]) appears at stage 3 in the center of the germ disc, and at stage 4 at its rim (S4A and S4B Fig). At stage 5, single cells spread from the center of the disc and scatter over the disc and the dorsal field (S4C–S4E Fig). These cells are likely endodermal and may contribute to the developing gut as they are expressed in very similar patterns as the endodermal marker genes serpent and hepatocyte nuclear factor 4 [44], (see also [42]). Later, FoxA-1 is expressed in broad segmental patches along the ventral midline, that in subsequent stages form a continues domain along the midline (S4F and S5 Figs). At stage 10 and subsequent stages, most of the expression of FoxA-1 disappears and transcripts only remain in the head and in the most posterior segments (S5G–S5I Fig).
Parasteatoda FoxA-2 is expressed later and first transcripts are detectable at around stage 8 in the stomodaeum and in segmental patches along the midline in anterior segments (Fig 2A–2C). At stage 9, all segments express FoxA-2 in the midline (Fig 2D–2F). In contrast to FoxA-1, expression of FoxA-2 does not disappear from central segments, but instead persists throughout the investigated developmental stages (Fig 2G–2I). Unlike Fox-A1, Fox-A2 is not expressed in the dorsal field (S4 Fig cf. panels F and G). S6 Fig shows DAPI staining of the embryos shown in Fig 2.
Expression of Parasteatoda FoxA-2 (A-I) and FoxC (J-O). In all panels, anterior is to the left, ventral view. Panels A, D, G, K, and M view of anterior with head. Panels B, E, H, L, and N view of middle part with walking limbs. Panels C, F, I, O view of opisthosoma. Arrows in panels K and M point to lateral expression in the head. The arrowheads in panels M and N point to faint expression at the ventral rim of the split germ band. Asterisks in panels K and L mark expression in the VNS. Filled circles in panels M-O mark expression at the base of the walking limbs. Abbreviations in Table 2. DAPI staining of the embryos shown in this figure are presented in S6 Fig.
Euperipatoides FoxA is first expressed in the mouth-anus (m-a) furrow and ventral tissue between the developing germ bands (S7A Fig). After closure of the m-a furrow, FoxA remains expressed in the mouth and the anus, as well as in tissue lining the ventral margins of the germ band proper. This expression persists throughout further development (S7B and S7C Fig). For further expression details, see also [18, 42].
FoxB.
Expression of FoxB genes in the here investigated arthropods and the onychophoran have recently been described in detail in [20, 45]. In all species, FoxB genes are expressed in the ventral sector of all appendages, except for the labrum of arthropods where expression is dorsal, and the onychophoran frontal appendages that do not express FoxB (S8 Fig). Additionally, FoxB is expressed in the ventral nervous system in all species (S8B, S8C, S8E, S8F, S8J, S8L, S8N and S8P Fig slim arrows). Early during development, in Tribolium both FoxB paralogs are expressed ubiquitously (S8A and S8D Fig). In Glomeris, first expression appears in the anlagen of the anal valves (S8G and S8H Fig). In the spider, FoxB is also expressed around the mouth/stomodaeum (S8K Fig).
FoxC.
Tribolium FoxC is first expressed in an anterior cap, which refines to expression in the mouth primordium (S9A and S9B Fig). When the germ band begins to elongate, FoxC is expressed ventrally in the head around the mouth (S9C Fig). This expression in principle remains throughout further development (S9D and S9F Fig). Additional expression appears in the proctodaeum, in the brain and in the VNS (the latter is marked by arrows in S9C–S9F Fig). After germ band retraction, FoxC is also expressed in the developing heart (cf. expression of FoxF, below) (S9F Fig, short arrow). Expression of FoxC in the head has also been reported previously by [46] Economou and Telford (2009).
At the blastoderm stage (stage 0), Glomeris FoxC expression is in the form of an anterior cap, very similar to the expression of other head-patterning genes in Glomeris [47] (S3E Fig). This domain transforms into expression surrounding the mouth and in the anterior head skeleton [cf. 48, 49] (S3F–S3H Fig). For the head patterning role of Glomeris FoxC, see also [47].
In Parasteatoda stage 7 embryos, expression of FoxC is in the anterior margin of the embryo (Fig 2J). Later, this domain refines into domains along either side of the mouth primordium, the pharynx and a pair of small dots (Fig 2K and 2M, arrow) in the pre-cheliceral region (Fig 2K and 2L). Segmental patches of expression are in the VNS (Fig 2K and 2L, asterisks), and at the base of the walking limbs (Fig 2M–2O, filled circles). Faint expression is at the ventral rim of the split germ band (Fig 2M and 2N, arrowheads). Some aspects of FoxC expression have also been described by [50]. S6 Fig shows DAPI staining of the embryos shown in Fig 2.
At stage 8, expression of Euperipatoides FoxC appears in the posterior of the head lobes and the anterior of the jaw-bearing segment (Fig 3A–3F, arrow and arrowhead respectively). This remains the only expression until stage 21 when mesodermal segmental patches appear along the anterior-posterior axis of the embryo (Fig 3G and 3H).
In all panels, anterior is to the left, ventral views, except panel D, lateral view, dorsal up. A´ and B´ represent DAPI staining of the embryos shown in A and B. In all panels, arrows point to expression at the posterior margin of the head lobes, and arrowheads indicate expression in the anterior of the jaw-bearing segment. Abbreviations in Table 2.
FoxD.
Tribolium FoxD appears in the form of two dots in the head lobes when germ band elongation has almost completed (Fig 4A). Later, segmental dots appear in an anterior to posterior progression in the VNS (arrows), and in the proctodaeum (Fig 4B and 4C).
Expression of Tribolium FoxD (A-C), FoxF (D-F) and FoxJ1 (G-I). In all panels, anterior is to the left, ventral view. Embryos are flat-mounted. Arrows in panels B and C mark expression in the VNS. Arrows in panels D and E point to the proctodaeum. Arrows in panels G and H mark dorsal expression. Arrowheads in panels D-F mark expression in the heart (dorsal tube). Asterisks in panel I mark dots of expression in dorsal tissue. Abbreviations in Table 2.
At stage 0, Glomeris FoxD is expressed in the form of an anterior cap (Fig 5A). Within this cap, a single stripe of enhanced expression appears; this stripe most probably represents expression in the primordium of the mandibular segment (Fig 5A, arrow). This assumption is based on the position of the stripe, the fact that we can follow the fate of the stripe over time, and the fact that the mandibular segment is often patterned first [51] (Fig 5B–5E). Shortly after formation of the first stripe, a second stripe appears at the posterior edge of the cap (Fig 5C). Then a third stripe appears that represents the ocular region (Fig 5D). At the same time, expression disappears from tissue between the ocular region and the mandibular stripe, and expression in the primordium of the proctodaeum appears (Fig 5D). Expression in the mandibular domain refines into a narrow but strong stripe (Fig 5E and 5F, asterisk in panel E). At stage 1.2, expression is still in the mandibular and the maxillary segment (Fig 5G and 5H). After this expression has disappeared, strong de novo expression appears in the VNS (Fig 5I–5K, arrows). At late developmental stages, FoxD is strongly expressed in the brain (Fig 5L). From stage 3 onwards, all tissue expresses FoxD weakly (Fig 5I–5L).
In all panels, anterior is to the left, ventral views. The arrow in panel B marks an appearing stripe of expression in the (likely) mandibular segment primordium. The asterisk in panel E marks the mandibular segment. Arrows in panels J and K mark expression in the VNS. Abbreviations in Table 2.
Parasteatoda FoxD is first expressed at sage 8.2 as a transverse stripe in the pre-cheliceral region (Fig 6A). Later, this domain splits into two domains in the developing brain (Fig 6B, 6D, 6G and 6H). At the same time, segmental patches of expression appear in the VNS of all segments except for the cheliceral segment (Fig 6B, 6C, 6E, 6F and 6I, arrows), and at the base of the walking limbs and pedipalps (Fig 6E and 6H, arrowheads). S10 Fig shows DAPI staining of the embryos shown in Fig 6.
In all panels, anterior is to the left, ventral views, except panels G, dorsal view and A, H, lateral view. Each row represents the same embryo, except for first row. Arrows point to expression in the VNS. Arrowheads point to expression at the base of the limbs. Abbreviations in Table 2. DAPI staining of the embryos shown in this figure are presented in S10 Fig.
Euperipatoides FoxD is first expressed in the posterior of the head lobes, but in a region slightly more anterior than that of FoxC (Fig 7A–7G). At stage 11, additional expression appears in the tips of the frontal appendages (Fig 7C–7G). At stage 16, weak mesodermal expression appears inside the jaws and the slime papillae (Fig 7E and 7F). At later stages, this expression is also present in the legs (Fig 7G and 7H).
In all panels, anterior is to the left. Panels A, B, and F, ventral view; panels C, E and G, lateral view, panel D, view of the head. A´/B´ represent DAPI staining of the embryos shown in A/B. Abbreviations in Table 2.
FoxF.
Tribolium FoxF is first expressed exclusively in the stomodaeum and the proctodaeum, although the most posterior tip of the embryo remains free from expression throughout development (not shown). Later, additional expression appears in the heart (Fig 4D–4F, arrowheads) (cf. expression of heart-patterning genes in [52]). Note the unspecific staining of the pleuropodia (pl) in panel F.
At stage 0.5, expression of Glomeris FoxF appears in a diffuse pattern in the trunk segments; the head remains free from expression (Fig 8A). At later developmental stages, this expression refines into segmental stripes that cover the middle of the ventral and dorsal segmental units of the trunk (Fig 8B–8D and S11A Fig). In late stage 3 embryos, a dot of expression appears on either side lateral to the mouth (Fig 5C and S11A Fig). Later, this tissue forms part of the foregut. Enhanced expression is inside the anal valves from stage 3 onwards (Fig 8B–8D and S11A and S11B Fig). At late developmental stages, FoxF is also expressed in the grooves between the developing tergites (Fig 8D, asterisks) [cf. 34, 53].
Expression of Glomeris FoxF (A-D), FoxJ1 (E-H) and FoxJ2 (I-L). In all panels, anterior is to the left, ventral views except panel D (lateral view, dorsal up). Asterisks in panel D mark expression in the tergite borders. Arrows in panel H mark dot-like expression in the head. Asterisks in panels K and L mark an area that is free of expression lateral in the head. Abbreviations in Table 2.
Parasteatoda FoxF-1 is first expressed at stage 8.2 in the form of faint patches in the second and third opisthosomal segments (Fig 9A–9C). Later, all opisthosomal segments express FoxF-1, mostly in dorsal tissue (Fig 9D–9I, arrows). Additional expression appears in the form of a faint stripe dorsal to the limbs of the prosoma and in the tail (Fig 9G and 9H). At late stages, almost the complete opisthosoma expresses Fox-F1 (Fig 9I). FoxF-2 is expressed in a subset of mesodermal cells in the pedipalps and the walking limbs in embryos of stage 10.2 (Fig 9J–9M, arrows) and later (not shown). S12 Fig shows DAPI staining of the embryos shown in Fig 9.
Expression of Parasteatoda FoxF-1 (A-I) and FoxF-2 (J-M). In all panels, anterior is to the left, ventral views, except panels G and M, lateral views, and H, dorsal view. Each row represents the same embryo, if not of different developmental stage. Arrows in panels D-H point to expression in the opisthosoma, and in panels J and K the legs. Abbreviations in Table 2. DAPI staining of the embryos shown in this figure are presented in S12 Fig.
Euperipatoides FoxF is first expressed anterior to the mouth, and in the form of a sharp band demarcating the anterior edge of the jaw-bearing segment (Fig 10A). A salt-and-pepper like expression is in the SAZ and newly formed segments (Fig 10A). At subsequent stages, expression is restricted to the dorsal edge of all segments (Fig 10B and 10C, asterisks), and some cells in the so-called dorsal-extraembryonic tissue (Fig 10B and 10C, arrows). At later stages this expression is not located at the dorsal edge of the embryo but in a position more ventrally, just dorsal to the position of the outgrowing limbs (Fig 10D–10F, arrow in panel D). From stage 20 onwards, additional expression appears in an anterior to posterior order along the ventral edge of the germ band (Fig 10E, 10G, arrowheads).
In all panels, anterior is to the left. Panels A and G, ventral view; panels B, D and E, lateral view; panels C and F, dorsal view. A´-D´ and G´ represent DAPI staining of the embryos shown in A-D and G. Arrow in panel D points to expression dorsally abutting the limb buds. Dashed line in panel D indicates dorsal margin of the germ band. Asterisks in panels B and C mark expression at the dorsal rim of the embryo; arrows in B and D point to expression in the dorsal extraembryonic tissue. Arrowheads in panels E and G mark expression in the ventral nervous system. Abbreviations in Table 2.
FoxG.
A detailed description of Tribolium FoxG-1 (slp) and FoxG-2 (slp2) has recently been published in [54]. The expression and function of slp has also been studied by [55].
In Glomeris, the appearance of segmental stripes in the post-blastoderm stage embryo is complex (S3I Fig). At later developmental stages, expression is in the brain (ocular region, oc), along the ventral midline, the limbs, in lateral segmental patches (arrows in panels K and L) and as transverse stripes in newly forming posterior segments (S3J–S3L Fig). The segmental expression pattern of Glomeris FoxG has also been described previously by [56, 57].
Parasteatoda FoxG is first expressed in the pre-cheliceral region at stage 8.1 (Fig 11A), and shortly later transverse stripes of expression appear in all segments (Fig 11B and 11C). This segmental expression persists throughout development (Fig 11D–11I). Later, expression appears in the labral region (Fig 11D) and in the developing heart (Fig 11G and 11H, arrows). S13 Fig shows DAPI staining of the embryos shown in Fig 11.
In all panels, anterior is to the left, ventral views, except panels G, lateral view and H, dorsal view. Each row (except first row, A-C) represents the same embryo. Arrow in panel B marks weak expression in the chelicerae segment. Arrows in panels G and H point to the heart. Abbreviations in Table 2. DAPI staining of the embryos shown in this figure are presented in S13 Fig.
Expression of Euperipatoides FoxG has been described by [39].
FoxH.
Among the investigated species, FoxH is only present in Euperipatoides where it is expressed inside the head lobes in early developmental stages (Fig 12).
In all panels, anterior is to the left, ventral views. A´/B´ represent DAPI staining of the embryos shown in A/B Abbreviations in Table 2.
FoxJ1.
Expression of Tribolium FoxJ1 appears by the end of germ band elongation in the form of two spots in the labrum, a diffuse pattern in the antennae and the walking limbs, a terminal domain in the labium, two spots in the first abdominal segment, and as defined spots dorsal in the second and third thoracic segment (arrows) (Fig 4G). Shortly after, additional dorsal expression appears in all abdominal segments (Fig 4H, arrow), the expression in the antennae becomes stronger, expression appears in the maxillae, and a spot of expression appears in the tip of the legs (Fig 4H). By the end of germ band retraction, the overall pattern is the same as described, with the exceptions that now several dorsal segmental spots are present (Fig 4I, asterisks), and that additional spots of expression appeared in the legs (Fig 4I).
Glomeris FoxJ1 is expressed ubiquitously at all stages, but there is enhanced dot-like expression in the ocular region at late developmental stages (Fig 8E–8H and S14B Fig).
From stage 10.2 onwards, Parasteatoda FoxJ1 is expressed in a number of single cells or clusters of cells in all limbs, including the labrum, spinnerets and book lungs (Fig 13A–13G). See figure legend for further information. S15 Fig shows DAPI staining of the embryos shown in Fig 13.
Expression of Parasteatoda FoxJ1 (A-G) and FoxL1 (H-L). In all panels, anterior is to the left, ventral views, except panels D, lateral view, F, dorsal view and G, lateral view. Arrow in panel A marks expression in the chelicera. Arrows in panels E and F point to expression in the spinnerets on opisthosomal segment 5. Asterisks in panels F and G mark a second dot of expression in O5. Arrows in panels H and I point to expression ventral to the base of the limbs. Arrowheads in panels I and L point to expression in the tail region. Abbreviations in Table 2. DAPI staining of the embryos shown in this figure are presented in S15 Fig.
We did not detect expression of Euperipatoides FoxJ1.
FoxJ2.
Fox J2 is missing in Tribolium. Glomeris FoxJ2 is expressed ubiquitously at all investigated developmental stages (Fig 8I–8L), except for late stages when transcripts are not seen in the lateral head region (Fig 8K and 8L, asterisks). Parasteatoda FoxJ2 is either not expressed in the investigated developmental stages, or is expressed ubiquitously at a very low level (data not shown). Euperipatoides FoxJ2 is first expressed in the frontal appendages (Fig 14A and 14B). Later, expression appears within the other appendages, in tissue dorsal to the limb buds, the anus (Fig 14C and 14D), and in a spot-like domain ventrally in the base of the limbs (Fig 14D, arrow).
Expression of Euperipatoides FoxJ2 (A-D) and FoxL (E-G). In all panels, anterior is to the left. Panels A, C and D lateral view. Panels B, and E-G ventral view. A´, D´ and E´-G´ represent DAPI staining of the embryos shown A, D and E-G. Arrow in panel D marks expression ventral to the base of the walking legs. Arrows and arrowheads in panels E-G point to expression at the posterior rim of the head lobes and the posterior pit respectively. Abbreviations in Table 2.
FoxK.
FoxK in Tribolium, Glomeris, Euperipatoides, and FoxK-1 in Parasteatoda are expressed ubiquitously at all investigated developmental stages (data not shown). Parasteatoda FoxK-2 either is not expressed in the investigated developmental stages, or is expressed ubiquitously at a very low level (data not shown).
FoxL1.
Tribolium FoxL1 is first expressed ubiquitously (Fig 15A), but by the end of germ band elongation, expression appears in the proctodaeum and in the form of weak spots in the VNS of the thorax and the abdomen, but not the head (Fig 15B–15E, arrows). By the end of germ band retraction, the proctodeal domain has split into one in the anus and one encircling the end of the hindgut. By this stage, expression is also in the Malpighian tubules (Fig 15D and 15E).
Expression of Tribolium FoxL1 (A-E) and FoxL2 (F-J). In all panels, anterior is to the left, ventral view. Embryos are flat-mounted, except embryos shown in panels A and F. Arrows in panels B, C, E and J point to expression in the VNS. Short arrow in panel D marks a ring of expression at the base of the Malpighian tubules. Note the unspecific staining of the pleuropodia in panels D, E, and J. Abbreviations in Table 2.
Glomeris FoxL1 is first expressed in a crescent-moon shaped domain anterior to the mouth primordium, and in the forming hindgut (Fig 16A). The anterior cells that express FoxL1 then sink in and form part of the stomodaeum; de novo expression appears in the brain (Fig 16B) and persists throughout further development (Fig 16C). At stage 3, diffuse expression in the posterior half of the embryo appears that is likely associated with endodermal tissue of the developing gut (Fig 16B and 16C, arrows)
Expression of Glomeris FoxL1 (A-D), FoxL2 (E-H), and FoxP (I-L). In all panels, anterior is to the left, ventral views (except panel H, lateral view). Arrows in panels B and C point to expression in the developing midgut. Arrowheads in panel G point to two domains of expression in a fusing dorsal segmental unit. Roman numerals mark expression in the dorsal segmental units (cf. Janssen 2011). The arrow in panel K points to expression in the VNS. The arrow in panel L points to expression in the last formed segment. Abbreviations in Table 2.
Parasteatoda FoxL1 is expressed in the stomodaeum (Fig 13H, 13J and 13K), in tissue ventral to the opisthosomal limb buds (Fig 13H and 13I, arrows) and the tail region (Fig 13I and 13L, arrowheads). S15 Fig shows DAPI staining of the embryos shown in Fig 13.
Euperipatoides FoxL1 is expressed in a small domain anterior to the mouth, the tissue posterior to the posterior edge of the head lobes (Fig 14E–14G, arrows), and in a horseshoe-like pattern in the posterior pit (the latter transforms into a simpler expression profile later during development) (Fig 14E–14G, arrowheads).
FoxL2
At early developmental stages Tribolium FoxL2 is either not expressed, or is expressed weakly and ubiquitously (Fig 15F). The first expression appears in the form of two transient segmental domains, one in the third abdominal segment, and one in the fifth (Fig 15G). By the end of germ band elongation, the abdominal expression domain in A3 has disappeared, and the one in A5 is very weak (Fig 15H). Segmental dots appear dorsal to the base of the labium, and the legs, and dorsally in the anterior abdominal segments (Fig 15H). Later, these dots are present in all abdominal segments (Fig 15I). Expression in the labial segment disappears at later developmental stages (Fig 15I and 15J). By the end of germ band retraction, weak expression in the VNS appears (Fig 15J, arrow).
Glomeris FoxL2 is exclusively expressed in the mesoderm of the dorsal segmental units of the trunk (Fig 16E–16H, indicated by Roman numerals). This expression is comparable with that of the myogenic marker nautilus (nau), although nau is expressed earlier than FoxL2 [cf. 58].
We isolated Parasteatoda FoxL2 from maternal cDNA but we could not detect any expression during ontogenesis. We were unable to detect expression of Euperipatoides FoxL2.
FoxM, FoxN14 and FoxN23
Expression of FoxM, FoxN14 and FoxN23 genes in the here investigated species has recently been described in [59].
FoxO
Tribolium FoxO is first expressed ubiquitously (Fig 17A and 17B), but when the germ band forms, expression is restricted to the anterior of the embryo proper, and at lower level in the anterior of the extraembryonic tissue. The expression in the embryo covers all anterior tissue with a sharp posterior border between the mandibular and maxillary segment (Fig 17C–17F, slim arrows). Later, this expression resolves into a complex pattern in the nervous system of the head (Fig 17G), that at later stages is also present in the entire embryo (Fig 17H).
Expression of Tribolium FoxO (A-H) and FoxP (I-K). In all panels, anterior is to the left, ventral view; except panel F, lateral view. In panel G, anterior is up. Embryos in panels E, H, J and K are flat-mounted. Long arrows in panels C and D mark the posterior border of strong expression in the head. Short arrows in panel C mark posterior border of expression in the extraembryonic tissue. Asterisks in panel H mark expression in the lateral tissue of the trunk segments. Filled circles in panel H mark dot-like expression in the VNS. Arrows in panels J and K point to expression in the VNS. Note the unspecific staining of the pleuropodia in panels H and K. Abbreviations in Table 2.
Glomeris FoxO is expressed ubiquitously (not shown). However, higher levels of expression are detectable in the labrum, and in the brain (S14C Fig).
Parasteatoda FoxO-1 is exclusively expressed in the dorsal field and the interface between the embryo proper and the so-called extraembryonic tissue around the head (Fig 18A–18I, arrows). Parasteatoda FoxO-2 is first expressed ubiquitously and in equal level in all tissue (not shown). From stage 10.1 onwards, stronger expression is visible in ventral segmental patches (Fig 18J–18L, arrows). In stage 10.2 embryos, ubiquitous expression disappears and strong expression is now in the ventral tissue of newly formed posterior segments (Fig 18O, asterisk and arrow), as well as in the mesoderm of walking limbs and pedipalps (Fig 18R), an ectodermal patch of expression at the dorsal base of the appendages (Fig 18N, arrow), two patches of expression in the brain (Fig 18M and 18P, arrowheads) and expression anterior to the mouth (Fig 18M and 18P, filled circles). At stage 13.1, posterior expression is restricted to the proctodaeal region (Fig 18Q). S16 Fig shows DAPI staining of the embryos shown in Fig 18.
Expression of Parasteatoda FoxO-1 (A-I) and FoxO-2 (J-R). In all panels, anterior is to the left, ventral views, except panels D, F, G, I, and R (lateral views), and H (dorsal view). Each row represents the same embryo, except panels G and H. Arrows in panels A, D, E and F point to expression in the interface between the embryo proper and the dorsal field. Arrow in panel J points to dot-like expression in the brain. Arrows in panels K and L point to expression in the VNS. Arrowheads in panels M and P point to lateral expression in the head lobes. Filled circles in panels M and P mark expression anterior in to the mouth. The asterisk in panel O points to strong expression in the VNS of nascent segments; the arrow in panel O points to weaker expression anterior to that. Arrowhead in N points to dot-like expression dorsal to the base of the walking legs. Abbreviations in Table 2. DAPI staining of the embryos shown in this figure are presented in S16 Fig.
Euperipatoides FoxO is first expressed ubiquitously, but stronger expression is in the posterior pit and anterior in the head lobes (Fig 19A). Later, expression is in the anterior of the head lobes (Fig 19B, arrow), in the posterior pit, the SAZ (where expression is in a strong transverse stripe, reminiscent of the expression of segmentation genes) (Fig 19B, arrowhead), and in a segmental pattern of weaker transverse stripes in the trunk segments (Fig 19C). In older (more anterior trunk segments) the segmental stripe-pattern disappears and only a dorsal segmental domain remains (Fig 19D and 19E). At stage 16, anterior trunk segments express FoxO ubiquitously, while in more posterior segments, the previously described dorsal pattern is still present; the posterior SAZ still expresses FoxO at a high level (Fig 19F).
Expression of Euperipatoides FoxO (A-F) and FoxP (G, H). In all panels, anterior is to the left. Panels A, B, E and H show ventral views; panels C, D, F, and G show lateral views. Arrow in panel B point to expression at the anterior rim of the head lobe. Arrowhead in panel B point to a segmentation gene like ventral stripe of expression in the last formed posterior segment. Abbreviations in Table 2.
FoxP
First, Tribolium FoxP is not expressed, or is expressed ubiquitously at a low level (Fig 17I). With the beginning of germ band retraction, strong expression appears in the brain, the stomodaeum (including the labrum), the proctodaeum and weakly in the VNS (arrows) (Fig 17J). This pattern remains throughout further development, but expression in the VNS becomes stronger (arrows), and expression in the developing Malpighian tubules appears (Fig 17K).
Glomeris FoxP is first expressed in the ocular region (Fig 16I–16L). Later it is also expressed in the form of dots in the mandibles, the maxillae, the antennae (albeit weakly), and the VNS (arrow in panel K) (Figs 16K and S14A Fig). After stage 6, the ventral tissue anterior to the SAZ expresses FoxP (Fig 16L, arrow), and faint expression is inside the anal valves (Fig 16L). Expression in the head appendages is restricted to mesodermal tissue; while most of the mesoderm in the mandibles and the maxillae expresses FoxP, expression in the antennae, the labrum and the walking limbs is restricted to a ventral and proximal portion of the mesoderm (S14A Fig).
At stage 9.2, Parasteatoda FoxP-1 is expressed in the anterior of the dorsal field (Fig 20A). In subsequent stages, this expression extends to the complete dorsal field (Fig 20B), and at stage 13.1, after dorsal closure, expression is in the dorsal of the opisthosoma (Fig 20C).
Expression of Parasteatoda FoxP-1 (A-C) and FoxP-2 (D-K). In all panels, anterior is to the left. Panels A and B show lateral view; panel C and L show dorsal views. All other panels show ventral views. Dotted lines mark the area of expression in the dorsal field. Arrowheads in panels G and H point to expression in the VNS. Arrows in panel J point to dot-like expression at the dorsal edge of the embryo. Abbreviations in Table 2. DAPI staining of the embryos shown in this figure are presented in S17 Fig.
Parasteatoda FoxP-2 is first expressed in the primordium of the mouth (Fig 20D and 20E). In subsequent stages, it is expressed in a large number of cells (or cell clusters) in the brain (Fig 20F), the VNS (Fig 20G and 20H, arrowheads) and segmental patches dorsal to the base of the limbs (Fig 20G and 20H, arrows), and later, in most cells of the central nervous system (Fig 20I–20K), except for newly formed posterior segments that express FoxP-2 later during development (Fig 20L). We did not detect any expression of FoxP-3. S17 Fig shows DAPI staining of the embryos shown in Fig 20.
Euperipatoides FoxP is expressed ubiquitously but in the limb buds and in the head lobes expression is stronger (Fig 19G). At stage 18, dots of expression appear in the walking limbs and the slime papillae (Fig 19H).
FoxQ1
FoxQ1 is not present in the here investigated arthropods, and we were unable to detect expression of FoxQ1 in Euperipatoides.
FoxQ2
Tribolium FoxQ2 is exclusively expressed in the head. First, expression in the form of two anterior domains is visible (S18A and S18B Fig). At the end of germ band elongation, these domains resolve into a complex pattern around the stomodaeum (S18C Fig). Expression is now also in the labral buds, the brain and around the mouth (S18C–S18E Fig). The expression and function of Tribolium FoxQ2 in head and brain development has been reported previously by [60].
Glomeris FoxQ2 is exclusively expressed in the head where it forms a complex pattern anterior and lateral to the mouth (S3M–S3P Fig). This expression corresponds to the tip of the labrum, the pharynx and a stripe and dot on either side of the labrum. Several aspects of Glomeris FoxQ2 expression have been reported by [61].
At stages 6/7 to 8.1, Parasteatoda FoxQ2 is expressed at the anterior margin of the early germ band (S19A and S19B Fig). Later, this domain refines into three patches of expression on either side of the mouth primordium (S19C–S19F Fig, asterisk, open circle, and filled circle). At stage 12, an additional pair of patches appears in the labrum (S19G and S19H Fig). The expression and function in labrum and nervous development have been described previously by [22].
Euperipatoides FoxQ2 is first exclusively expressed ventrally in the head lobes, anterior to the mouth (S20A and S20B Fig). At later developmental stages, faint expression appears in a small domain ventral of the eyes (S20C and S20D Fig, asterisks) and between the base of the jaws and the slime papillae (S20C and S20D Fig, arrowheads). [61] has also described several aspects of the Euperipatoides FoxQ2 expression profile.
FoxT (syn. fd3F)
Tribolium FoxT is only expressed in late developmental stages (Fig 21). Dots of expression are in the limbs, and in dorsal tissue along the body, similar as described for FoxJ1 and FoxL2.
Panel A ventral view, panel B lateral view and panel C close-up on head, dorsal view. Note the unspecific staining in the pleuropodia in panels A and B. Abbreviations in Table 2.
We did not find this Fox gene in Glomeris and Parasteatoda, and we did not detect expression of FoxT in Euperipatoides.
Discussion
Panarthropod Fox genes
The phylogeny and gene content of panarthropod Fox genes have recently been discussed in [28]. According to this analysis, two classes of Fox genes appear to have been lost in Panarthropoda, FoxE and FoxM. Additionally, FoxH has been lost in Arthropoda. A recent analysis, however, claims to have identified a FoxM gene in Drosophila (i.e. CG32006), a gene that we and others believe is a FoxJ1 ortholog (cf. [62] with [27, 28]). Gene expression analysis of CG32006/FoxJ1 genes supports this interpretation (discussed below). Another potential loss in Arthropoda may concern FoxQ1, but [28] reported a potential FoxQ1 gene in a scorpion (Fig 1). The onychophoran Euperipatoides possesses a large set of Fox genes with single members of all expected classes including FoxH. Additionally, Euperipatoides possesses an orphan gene that recently has been described as a potential FoxM class gene [59]. In the analyses performed by [28], this gene clustered with FoxM genes from other animals, albeit with low support. The water bear Ramazzottius varieornatus, however, lacks the otherwise conserved genes FoxD, FoxJ1, FoxJ2/3 and FoxN23, and also possess no FoxM and no FoxH (unlike the onychophoran). Overall, the tardigrade thus appears to have retained a much less well conserved set of Fox genes than the onychophoran.
For a pair of Drosophila Fox genes that were long considered orphans (fd3F and Crg-1), [14, 27, 28, 63] identified orthologs in most of the investigated insect species, the water flea Daphnia, a scorpion (albeit with weaker support), and the onychophoran Euperipatoides (Fig 1). These genes are considered to form a separate group of Fox genes named FoxT [27, 28]. The unique expression of FoxT (fd3F and Crg-1) genes support the hypothesis that they form a separate group of Fox genes.
On the function of Fox genes in animals
FoxA–A conserved factor of metazoan gut development. The FoxA ortholog forkhead, the first-identified and founding member of the Fox gene family, is an important player in the development of the ectodermal foregut and hindgut, but also the endodermal posterior midgut and the Malpighian tubules, in the fly Drosophila [64–66]. In other arthropods like for example the beetle Tribolium [41], the millipede Glomeris [42], the spider Parasteatoda [43], and the onychophoran Euperipatoides [42], fkh is also likely involved in gut development (ectodermal fore- and hindgut and endodermal midgut). Expression and thus implied function of fkh in the Malpighian tubules appears to be restricted to insects (or possibly Pancrustacea) because in the millipede Glomeris, fkh is not expressed in the Malpighian tubules [42]. In other ecdysozoans such as the nematode worm Caenorhabditis and the priapulid worm Priapulus, the function in gut development appears to be conserved as well [67–69]. Outside Protostomia, a general function of FoxA in gut development appears to be conserved in lophotrochozoans [7, 10, 70–77].
FoxB–A factor of dorsal-ventral body and appendage patterning. In Drosophila, FoxB orthologs are expressed in the fully extended germ band stage embryo, in neuroblasts and sensory neurons [15]. A recent study showed that FoxB is expressed in conserved patterns in arthropods including Drosophila and an onychophoran [20]. FoxB is expressed in the ventral sector of the limbs in all panarthropod species, where it is likely involved in dorsal-ventral limb patterning, as functional data from the spider Parasteatoda suggest [20]. In addition, FoxB is also involved in the transformation of the early germ disc into the bilateral germ band, and thus in dorsal-ventral body patterning [45]. Expression data on FoxB in other ecdysozoans is restricted to Caenorhabditis (syn. lin-31) where it is inter alia involved in vulva-development. Interestingly, during this process lin-31 expression is restricted to a subset of ventral cells, and without the input of lin-31, these cells randomly either contribute to the vulva or not, indicating that lin-31 acts as a binary genetic switch [78, 79]. FoxB/lin-31 therefore likely acts as a ventral factor. In echinoderms, FoxB (syn. fkh1) is expressed in the mesenchyme and is involved in gut development. Strongest expression is at the oral (ventral) side of the developing embryo [80–82]. In the hemichordate Saccoglossus, FoxB is expressed in a complex pattern, but it is striking that FoxB is asymmetrically expressed on one side of the blastopore [7]. In vertebrates, FoxB (syn. fkh5) is expressed in the dorsal ectoderm of the organizer [83, 84]. Given that the dorsal-ventral axis is reversed in chordates vs protostomes (reviewed in [85]), this means that also here FoxB is a marker of (ancestrally) “ventral” tissue. These patterns are comparable to the expression along the ventral ectoderm in panarthropod limbs and the ventral cells in Caenorhabditis, and therefore, it is possible that FoxB is a general discriminator of dorsal versus ventral tissue. If so, this function dates back to the last common ancestor of panarthropods and chordates, the urbilaterian.
FoxC–A conserved factor of anterior gut and mouth development. In Drosophila, FoxC (syn. crocodile/croc) is involved in the development of head structures such as the inner head skeleton [86], and this function appears to be conserved in panarthropods, including the species investigated in this study (see also [17, 46, 47, 50, 87]. In the annelid Capitella, FoxC is also dominantly expressed in mesodermal structures in the head and around the foregut [9]. Similarly, in the leech Helobdella austinensis, FoxC is expressed in the musculature associated with the developing proboscis [73]. In a brachiopod larva, FoxC is expressed in the anterior of the archenteron and associated structures [88]. In the hemichordate, FoxC is involved in the development of anterior structures such as the proboscis and the anterior mesoderm [7]. In vertebrates, FoxC genes are expressed in the mesoderm and are involved in head development as well, including the development of pharyngeal structures [89] (and references therein). In the cnidarian Nematostella that like all cnidarians lacks mesoderm, FoxC is expressed in the pharyngeal endoderm and the first-developed mesenteries, but not the body endoderm or the other mesenteries [90]. Given that the mesoderm may have evolved from the endoderm in the diploblast ancestor [91], this expression may be homologous to that of anterior mesodermal structures in bilaterian animals.
Altogether, these expression patterns imply a specific function of FoxC genes in head mesoderm development that likely dates back to the last common ancestor of all eumetazoan animals.
FoxD–A conserved factor of ecdysozoan nervous system patterning. Drosophila FoxD is expressed in a subset of procephalic neuroblasts, some neuroblasts in the VNS, and sensory organs in the trunk and in the brain [15, 92]. This pattern is conserved in Tribolium, Glomeris, and Parasteatoda, where FoxD is strongly expressed in the brain and the VNS. In the onychophoran, however, there is only expression in the brain, but not along the VNS, suggesting that this latter aspect of FoxD is restricted to Arthropoda.
In Caenorhabditis, FoxD (syn. unc-130) [16] is involved in axon guidance and the specification of neuronal tissues [93, 94], as well as in dorsal-ventral patterning of the postembryonic mesoderm [95, 96]. In other protostomians such as brachiopods and annelids, FoxD is expressed in both mesodermal and ectodermal derivatives [88, 97]. In deuterostomes like echinoderms, FoxD appears to be predominantly expressed in ectodermal tissue [8, 98]. In ascidians, FoxD is involved in the patterning of mesodermal and endodermal tissue, in notochord induction, and in the patterning of the animal-vegetal body axis [99–101]. In the hemichordate Saccoglossus, and the cephalochordate Branchiostoma, FoxD is expressed in mesodermal tissues [7, 102]. In Nematostella, FoxD is first expressed at the aboral pole suggesting a role in patterning the oral-aboral axis. Later it is expressed at the base of the tentacles [90].
In summary, this suggest that FoxD played a role in both ectoderm and mesoderm patterning in the last common ancestor of at least Bilateria, and that lineage-specific losses of mesodermal or ectodermal expression/function happened frequently in different evolutionary lineages (see also [88]). In panarthropods, and possibly in ecdysozoans as a whole, FoxD appears to be involved in the development of the nervous system.
FoxF–A conserved factor of visceral mesoderm development. In Drosophila, FoxF is involved in the formation of the visceral mesoderm and the midgut [103–106]. Expression in Tribolium, Glomeris, Parasteatoda, and Euperipatoides appears to be mainly in mesodermal tissue of the trunk suggesting that the function of FoxF in the patterning of the visceral mesoderm, or at least part of it, is conserved in Panarthropoda. Data from other ecdysozoans are restricted to Caenorhabditis. Here, the single FoxC/FoxF gene (syn. Let-381) is involved in the development of non-muscle mesodermal tissue [107, 108]. In other protostomes like brachiopods, planarians and molluscs the function in visceral muscle development appears to be conserved [9, 88, 109], and so is this function in deuterostomes such as echinoderms, ascidians, hemichordates and vertebrates (e.g. [7, 110–112]).
Summarized, our current knowledge on FoxF genes strongly suggests a conserved function in visceral mesoderm development in bilaterian animals. Interestingly, there is no clear ortholog of FoxF in cnidarians which could be correlated to the lack of clear-cut mesoderm in these animals (e.g. [113, 114]).
FoxG–A conserved factor in arthropod segmentation, and bilaterian brain and ciliary nervous system development. The Drosophila FoxG orthologs sloppy-paired 1 (slp1) and sloppy-paired 2 (slp2) play redundant but essential functions in the segmentation gene cascade where they act as segment-polarity and pair-rule genes [115–117]. The segmentation gene function of FoxG has been investigated in various other arthropods and an onychophoran, suggesting that it is conserved in arthropods but not onychophorans (e.g. [55, 39, 55, 118–120]). In Drosophila, slp1, but not slp2, also acts as an important factor of early head development [121]. Interestingly, our recent phylogenetic analysis revealed that the previously identified fd19B gene [14] represents a third FoxG-class gene in Drosophila [28, 62] (Fig 1 and S1 Fig). Its expression pattern suggests that it may contribute to the function of slp1 in head development.
In Caenorhabditis, FoxG (syn. fkh-2), is involved in the development of a subset of ciliary neurons [122]. In annelid larvae, FoxG is expressed in the brain and a subset of cells of the ciliated bands that are in close proximity to the locomotory cilia [123, 124]. In a planarian, it is expressed in the brain [125]. In echinoderms, FoxG is expressed in the ciliary bands where it is likely involved in patterning the underlying nervous system [8, 126]. In hemichordates, FoxG is expressed in the anterior of the developing embryo that harbors the brain and, at earlier developmental stages, it is expressed in close proximity to the ciliated band [7]. In cephalochordates, FoxG is involved in brain development including the development of nerves that are associated with ciliated sensory receptors [127]. In vertebrates, FoxG is involved in the development of the telencephalon (e.g. [128]).
The available data suggest that the role of FoxG genes in segmentation and head development is restricted to arthropods, and that the ancestral function of FoxG was likely in brain development including the development of the nervous system associated with ciliary cells.
FoxH. In the onychophoran, FoxH is expressed transiently in the head lobes where the brain will form. Data on FoxH from other animals is extremely scarce. In chordates, it appears to be involved in the development of left-right asymmetries of the main body axis [129].
FoxJ1 –A conserved factor of motile cilia development. Primary cilia (i.e non-motile cilia) and motile cilia are known from a wide range of animals. In ecdysozoans, however, only the sperm and the chordotonal organs (bipolar neurons) possess motile cilia (e.g. [130]). FoxJ1 appears to be a master gene of motile cilia development as it is not only needed but also sufficient to induce motile cilia development [131, 132]. Both primary cilia and motile cilia are under control of another transcription factor, the Regulatory factor X (Rfx) (e.g. [133]). In Drosophila and other ecdysozoans, the function of Rfx in primary cilia development appears to be conserved (e.g. [134, 135]). The suggested loss of FoxJ1 in ecdysozoans as a regulator of motile cilia was therefore not very surprising [24].
Recent studies, however, showed that the previously uncharacterized Drosophila forkhead-box gene CG32006 likely represents a FoxJ1-class gene, and that FoxJ1 genes are present in at least most of Panarthropoda [27, 28]. We found gene expression data of CG32006/FoxJ1 in the Berkeley Gene Expression Patterns database (BDGP, [136, 137]). Drosophila FoxJ1 is expressed in the form of two distinct domains in the anterior head and numerous small dots of expression along the body, a pattern that could correlate with the development of bipolar neurons. In this context, it is interesting to note that Rfx-dependent genes that are supposed to be expressed in all ciliated cells (e.g. CG31036 [134]) are indeed expressed in a larger number of cells (or cell clusters) than FoxJ1 (CG32006) (see BDGP), which would be in line with a potentially exclusive function of FxoJ1 in the development of motile cilia.
The here reported expression profiles of FoxJ1 in Tribolium and Parasteatoda are comparable with those of Drosophila FoxJ1. Detection of FoxJ1 transcripts in the “embryonic” transcriptome of Euperipatoides suggests late expression in developmental stages that have been included in mRNA extraction for transcriptome sequencing, but that are too late in development to work in whole mount in-situ hybridization experiments. Note in this context that expression of another potentially conserved motile cilia-specific Fox gene, FoxT, was not detected in the investigated embryonic stages of the onychophoran either (discussed below).
Data on FoxJ expression outside Ecdysozoa support the idea that FoxJ1 is a conserved factor of motile cilia development: In annelids, FoxJ1 is expressed in association with ciliary and sensory cells [138], and in a sea urchin, it is expressed in the area of the apical plate and the ciliated bands [8]. In hemichordates, FoxJ1 is also involved in the development of the ciliated band and the apical organ [7]. In vertebrates, FoxJ1 is associated with the development of ciliated cells (e.g. [131, 139, 140]). In cnidarians such as Nematostella, FoxJ1 is expressed in the ciliated apical organ [141], and it has been shown that FoxJ1 is also present in the earliest animals, sponges and choanoflagellates [142, 143].
In summary, as previously suggested by [132], FoxJ1 appears to be a conserved regulator of motile cilia cell development, and as we show here, this may even be the case in arthropods.
FoxJ2 (syn. FoxJ2/3). The expression patterns of Euperipatoides, Glomeris, and Parasteatoda are diverse and thus do not allow speculation on conserved functions. In the latter two species, FoxJ2 is expressed ubiquitously, and thus not very informative. Other data on FoxJ2 expression are scarce. In Saccoglossus expression of FoxJ2 could not be detected [7]. In the frog Xenopus, FoxJ2 is first expressed ubiquitously, but at later developmental stages it is expressed in the notochord and the ventral region of the neural tube [144]. In mouse, FoxJ2 regulates meiosis in spermatogenesis [145]. FoxJ2 is also involved in some forms of cancer (e.g. [146]). In vertebrates, FoxJ3 appears to be a neurogenic factor [147], and this is also the case in the cnidarian Hydra, where FoxJ3 appears to be involved in neurogenesis [148].
FoxK–A potentially conserved factor of cell cycle control. FoxK is present and expressed ubiquitously in all investigated panarthropod species, including Drosophila [136, 137]. Functional studies have shown that at later developmental stages, FoxK is involved in the formation of the midgut in Drosophila [149]. In Saccoglossus, expression of FoxK is ubiquitous in the ectoderm, although expression is weaker in the ciliary band [7]. It has recently become clear that FoxK class genes are involved in cell cycle control and cancer (reviewed in [150]). Although information on FoxK expression and function is scarce, it appears likely that it is involved in cell metabolism and/or cell cycle control (cf. ubiquitous expression of FoxN class genes and their function in cell cycle control).
FoxL1 –A potentially conserved factor of gut (and associated structures) development. Drosophila FoxL1 is expressed in a posterior and ventral region of the blastoderm that then invaginates to form part of the posterior mesoderm. Later, pairs of segmental clusters of FoxL1-expressing cells appear in the trunk [15, 151]. It has been shown that FoxL1 plays a role in organ placement, and that in knock-out fly embryos, various organs like the germ cells and the Malpighian tubules fail to position correctly [151]. In Tribolium, the expression of FoxL1 is conserved in the developing hind and midgut and in segmental dots in the trunk. In Glomeris, Parasteatoda and Euperipatoides, we find a comparable early posterior domain of expression that demarcates the hindgut. The segmental expression, however, is not present in Glomeris, and the anterior expression in the brain and the mouth/pharynx seen in these species is not present in Drosophila and Tribolium. In Saccoglossus and the shark Scyliorhinus, FoxL1 is expressed in the developing gill slits [7, 89]. In vertebrates, FoxL1 is an important component of gut development [152, 153].
It appears that FoxL1 is a conserved factor in gut development, including the development of associated structures such as the pharynx, Malpighian tubules, and gill slits.
FoxL2
FoxL2 is absent in Drosophila but is present in most other panarthropods (Fig 1). However, in the spider and the onychophoran, we could not detect expression. In Tribolium and Glomeris, FoxL2 is expressed late during development and is mainly restricted to dorsal segmental tissue of the trunk segments. In the planthopper Nilaparvata, expression of FoxL2 is female-specific and has a function in chorion development [27, 154]. Besides the potentially conserved function of FoxL2 in egg-development, we assume that there is a conserved function of this gene in patterning dorsal tissue (possibly muscles) in at least mandibulate arthropods.
In the leech Helobdella, FoxL2 is expressed in developing muscle tissue as well [73]. In the echinoderm Strongylocentrotus, FoxL2 is not detectable or is expressed ubiquitously at low levels early during embryogenesis [8] (their Supporting information). In Saccoglossus, FoxL2 is present, but transcripts could not be detected [7]. In vertebrates, FoxL2 is a known factor of female gonadogenesis (reviewed in [155]), a function that could be conserved in the oyster Crassostrea [156]. Interestingly, in both groups of animals there is an anti-sense transcript of FoxL2 that is likely involved in the regulation of FoxL2 sense transcripts [157, 158]. However, we did not detect expression using sense-probes neither for Glomeris nor for Tribolium FoxL2 (data not shown). In the sponge Suberites expression is ubiquitous [13].
FoxM, FoxN14, and FoxN23 –A trio of cell cycle controlling genes. FoxM appears to be lost in arthropods (Fig 1). In the onychophoran, FoxM, FoxN14 and FoxN23 all are expressed in a complex dynamic pattern, suggesting a function in cell cycle control [59]. In other arthropods, expression of FoxN genes is ubiquitous [59], a pattern that is in line with a function in mitotic cells and thus cell cycle control. However, the available panarthropod data suggest that the situation in Drosophila, where FoxN genes are differentially expressed in various tissues, is derived [159–162].
The role of FoxM, and FoxN genes in cell cycle control is also conserved in vertebrates (reviewed in [163, 164]) [165–167], suggesting that a function of these genes in controlling the cell cycle is conserved among at least Bilateria.
FoxO. Expression profiles of panarthropod FoxO orthologs are diverse. In Drosophila, FoxO is expressed maternally, but soon after fertilization, transcripts disappear until at stage 11 when de novo expression appears in ectodermal and endodermal tissue. Expression levels in ventral tissue of the trunk and the head are low except for the labrum that strongly expresses FoxO [14]. In Tribolium, zygotic expression is mainly restricted to the head region and later in the developing brain and nervous tissue in the head. In Glomeris, expression is ubiquitous. Of the two Parasteatoda FoxO orthologs, FoxO1 is expressed in the dorsal field, and FoxO2 is expressed in complex patterns during development. Finally, onychophoran FoxO is expressed in a complex pattern as well; some aspects of its expression may be conserved between the spider and the velvet worm.
In Caenorhabditis, FoxO (syn. daf16) is a mediator of dauer formation (halting development) and aging [168, 169], mediates insulin-like metabolic signaling and stress resistance, and is involved in learning, memory, and regeneration [170], many of the functions that are conserved in Drosophila, other insects such as the silkworm Bombyx [171–173] and mouse (reviewed in e.g. [174]). In Saccoglossus, FoxO was not detectable in early development [7]. In Hydra, FoxO is involved in the regulation of stem cell proliferation and antimicrobial peptides that are components of the immune system and the microbiome [175, 176]. The only expression data from lophotrochozoan species come from the leech Helobdella austinensis where its two FoxO genes both are expressed in complex patterns [73], and the planarian Schmidtea mediterranea where the gene appears to be expressed ubiquitously [62].
Altogether, FoxO genes appear to represent important and conserved factors in regulating animal metabolism. This is in line with the often-ubiquitous patterns of FoxO during development.
FoxP—A conserved factor of bilaterian nervous system development. In Drosophila, FoxP is expressed in the yolk cytoplasm as well as in the central nervous system where it starts with the occurrence of segmental groups of FoxP-expressing cells along the ventral midline. Later, the complete central nervous system expresses FoxP [14]. Functional studies have shown that FoxP indeed is needed for developmental processes in the nervous system (e.g. [177–179]). In the honey bee Apis mellifera, and other bees, FoxP is also expressed in the brain [180, 181]. This pattern is conserved in the here investigated arthropods and in the onychophoran. In all species, at least one paralog is expressed in the brain and the VNS. In the spider, one of the two paralogs, FoxP1 is expressed in the dorsal field. Most probably this pattern represents a neo-functionalization after the duplication, whereas the second paralog, FoxP2 fulfils the ancestral function in nervous system patterning. Although the pattern is less clear in the onychophoran, FoxP is strongly expressed in the brain and in the region of the VNS. The function of FoxP in nervous system patterning is thus likely conserved in Panarthropoda.
In Saccoglossus, FoxP is predominantly expressed in ectodermal tissue, suggesting that also here, FoxP may be involved in nervous system patterning [7], and in vertebrates, FoxP is known to be a key player of nervous system development (e.g. [182–184]), suggesting that FoxP is a universal factor of bilaterian nervous system development. FoxP genes have been identified in cnidarians and even sponges [142, 185], but expression or functional data are not available leaving the question open of whether the suggested function of FoxP as a neuronal gene may date back even beyond Bilateria.
FoxQ1 –A conserved factor of pharynx development. Although we identified a FoxQ1 gene in the onychophoran Euperipatoides (Fig 1) [28], we could not detect expression in the developmental stages that we investigated. In urochordates, hemichordates, cephalochordates, and vertebrates, FoxQ1 is specifically expressed and functions in the development of pharyngeal structures (e.g. [7, 24, 144, 186]). Interestingly, in an annelid, FoxQ1 is expressed in the pharynx as well [9], suggesting that the ancestral function of FoxQ1 in development is restricted to the development of the pharynx.
FoxQ2 –A highly conserved factor of anterior development. FoxQ2 is a factor of the so called anterior gene regulatory network (aGRN) that appears to be highly-conserved in all Bilateria, and even diploblasts (e.g. [123, 138, 141, 187]). Consequently, in all hitherto investigated arthropods, FoxQ2 is expressed in the anterior of the developing embryo including the anlagen of the pharynx and the anterior procephalic neuroectoderm [14, 22, 60, 61, 188, 189]. Given the conserved expression patterns of other anterior patterning genes such as six3 and orthodenticle (otd) the anterior patterning GRN as a whole, or at least key-components of it, appear to be conserved in panarthropods (e.g. [47, 61, 188, 190–192]), and indeed all groups of animals (e.g. [190, 193–195]), although, surprisingly, FoxQ2 has been lost in placental mammals (e.g. [5, 24]).
FoxT–A potentially conserved factor of hexapod chordotonal sensory cell development. A new class of Fox genes, FoxT, was recently identified [27, 28]. In Drosophila, two genes belong to this class, fd3F and Crg-1 (Fig 1 and S1 Fig). fd3F is first expressed ubiquitously, but from stage 12 onwards expression is exclusively in cell clusters along the ventral and lateral side of the embryo [14, 63, 196]. These cell clusters correspond to chordotonal (Ch) sensory organs and their precursors, and it has been shown that fd3F regulates specification of this group of ciliated neurons, while the other group of ciliated neurons, the external sensory (ES) neurons, do not express fd3F [63, 196]. The function of fd3F is thus similar to that of another Fox gene, FoxJ, and it has been suggested that fd3F may represent a highly-derived FoxJ-class gene [63]; recent phylogenetic analyses, however, do not support this idea (discussed above) [27, 28].
Our data suggest that the function of fd3F is conserved in at least insects, because the expression pattern of Tribolium FoxT/fd3F are very similar to that in Drosophila. We could not detect specific expression of Euperipatoides FoxT/fd3F, which could be explained by the relatively late development of Ch neurons (cf. expression in insects), and gene expression studies in late stages of onychophorans are problematic.
Drosophila Crg-1 is expressed in the adult head, and is involved in steering the circadian rhythm of the fly [197]. [28] suggested that fd3F and Crg-1 are the result of a duplication event of FoxT in Drosophila.
The single FoxT-type gene of the planthopper Nilaparvata appears to be exclusively expressed in the testis of late male nymphs and adult males [27]. This finding could explain why we were not able to detect expression of FoxT in embryos of the onychophoran Euperipatoides. The lack of FoxT earlier in the development of Nilaparvata, however, suggests that the pattern (and thus function) of FoxT reported in Drosophila and Tribolium may be restricted to holometabolous insects.
Supporting information
S1 Fig. The complement of panarthropod Fox genes investigated in this paper and of the model arthropod Drosophila melanogaster.
Each box indicates one paralog of a given Fox-class gene. Horizontal black bars indicate gene loss.
https://doi.org/10.1371/journal.pone.0270790.s003
(TIF)
S2 Fig. Expression of Tribolium FoxA.
In all panels, anterior is to the left. A Ventral view. B Lateral view. C Dorsal view. D Dorsal view of posterior end of embryo. The short arrows in A-C indicate expression laterally in the head lobes. The long arrow in A marks expression along the ventral midline. Asterisks mark unspecific staining of the pleuropodia. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s004
(TIF)
S3 Fig.
Expression of FoxA (A-D), FoxC (E-H), FoxG (I-L), and FoxQ2 (M-P). In all panels, anterior is to the left, ventral views (except panels D and L, ventral lateral). The arrow in panel D point to expression in the VNS. The asterisk in panel I marks the mandibular segment. Arrows in panels K and L mark lateral dots of expression. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s005
(TIF)
S4 Fig.
Early expression of FoxA-1 (A-F) and comparison of expression of FoxA-1 (F) and FoxA-2 (G) in the dorsal field. Note that FoxA-1, but not FoxA-2 is expressed in the dorsal field. The x in panel A marks the center of the germ disc that expresses FoxA1. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s006
(TIF)
S5 Fig. Expression of Parasteatoda FoxA-1.
In all panels, anterior is to the left, ventral view. Panels A, D and G, view of anterior with head. Panels B, E and H view of middle part with walking limbs. Panels C, F, and I view of opisthosoma. Asterisks in panel G mark expression in the chelicerae. A´-I´ represent DAPI staining of the embryos shown in A-I. Each row (e.g. A-C) represents the same embryo. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s007
(TIF)
S6 Fig. DAPI staining of the embryos shown in Fig 2.
Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s008
(TIF)
S7 Fig. Expression of Euperipatoides FoxA.
In all panels, anterior is to the left, ventral views, except panel B, lateral view, dorsal up. A´-C´ represent DAPI staining of the embryos shown in A-C. Arrows in panels B and C mark expression along the ventral margin of the embryo proper. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s009
(TIF)
S8 Fig. Expression of FoxB.
Expression of Tribolium FoxB1 and FoxB2 (A-F), Glomeris FoxB (G-J), Parasteatoda FoxB (K-M), and Euperipatoides FoxB (N-P). In all panels, anterior is to the left (except panel M, ventral to the left). All panels represent ventral views (except panels M, N and P, lateral views). Narrow arrows in panels C, E, F, L, N and P point to the ventral nervous system. The asterisks in panels C and F mark unspecific signal in the pleuropodia. The arrow in panel J points to the midline. The arrow in panel K points to expression around the mouth (stomodaeum). The asterisk in panel K marks expression in the posterior end of the embryo. The arrow in panel O points to expression in the ventral tissue of the appendage. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s010
(TIF)
S9 Fig. Expression of Tribolium FoxC.
In all panels, anterior is to the left, ventral view. Embryos in D-F are flat-mounted. Long arrows in panels C-F mark expression in the VNS. Short arrow in F points to expression in dorsal tissue that could contribute to the heart. Asterisks in F mark unspecific staining in the pleuropodia. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s011
(TIF)
S10 Fig. DAPI staining of the embryos shown in Fig 6.
Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s012
(TIF)
S11 Fig. Additional aspects of Glomeris FoxF expression.
Anterior is to the left, ventral views. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s013
(TIF)
S12 Fig. DAPI staining of the embryos shown in Fig 8.
https://doi.org/10.1371/journal.pone.0270790.s014
(TIF)
S13 Fig. DAPI staining of the embryos shown in Fig 9.
Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s015
(TIF)
S14 Fig.
Expression of Glomeris FoxP (A), FoxJ1 (B) and FoxO (C), additional aspects. Anterior views. Arrowhead in panel B points to dot of expression in the lateral head. Arrowhead in panel C points to expression in the labrum. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s016
(TIF)
S15 Fig. DAPI staining of the embryos shown in Fig 13.
Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s017
(TIF)
S16 Fig. DAPI staining of the embryos shown in Fig 18.
Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s018
(TIF)
S17 Fig. DAPI staining of the embryos shown in Fig 20.
Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s019
(TIF)
S18 Fig. Expression of Tribolium FoxQ2.
In all panels, anterior is to the left, ventral view. Embryos are flat-mounted, except embryo shown in panel A and E. The out-of-focus signal in the center of the embryo shown in panel E is in the pleuropodia that stain unspecific. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s020
(TIF)
S19 Fig. Expression of Parasteatoda FoxQ2.
In all panels, anterior is to the left, anterior view, except panels B, lateral view. A´-H´ represent DAPI staining of the embryos shown in A-H. In all panels, asterisks, filled circles and open circles mark corresponding domains of expression during development. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s021
(TIF)
S20 Fig. Expression of Euperipatoides FoxQ2.
In all panels, anterior is to the left. Panels A and B, ventral view; panel C, lateral view, dorsal up; panel D, dorsal view. A´-D´ represent DAPI staining of the embryos shown in A-D. Asterisks in panels C and D mark faint expression ventral to the eyes. Arrowheads in panels C and D point to expression in the interface between jaws and slime papillae. Abbreviations in Table 2.
https://doi.org/10.1371/journal.pone.0270790.s022
(TIF)
Acknowledgments
We are thankful for support in the form of clones, cDNAs and embryos from Mathias Pechmann, Parasteatoda, (University of Cologne, Germany), Alistair McGregor, Parasteatoda, (Oxford Brookes University, England) and Gregor Bucher, Tribolium, (University of Göttingen, Germany). We gratefully acknowledge the support of the New South Wales Government Department of Environment and Climate Change by provision of a permit SL100159 to collect onychophorans at Kanangra-Boyd National Park. We thank Glenn Brock, David Mathieson, Robyn Stutchbury and especially Noel Tait, for their help during onychophoran collection. Whole-mount in-situ hybridization experiments were partially performed under the supervision of RJ during the “Evolution and Development” course at Uppsala University in 2016; course no. 1BG391.
References
- 1. Weigel D, Jäckle H. The fork head domain: a novel DNA binding motif of eukaryotic transcription factors? Cell 1990;63: 455–456. pmid:2225060
- 2. Hannenhalli S, Kaestner KH. The evolution of Fox genes and their role in development and disease. Nat Rev Genet 2009;10: 233–240. pmid:19274050
- 3. Jackson BC, Carpenter C, Nebert DW, Vasiliou V. Update of human and mouse forkhead box (FOX) gene families. Hum Genomics 2010;4: 345–352. pmid:20650821
- 4. Wotton KR, Shimeld SM. Analysis of lamprey clustered Fox genes: insight into Fox gene evolution and expression in vertebrates. Gene 2011;489: 30–40. pmid:21907770
- 5. Yu JK, Mazet F, Chen YT, Huang SW, Jung KC, Shimeld SM. The Fox genes of Branchiostoma floridae. Dev Genes Evol 2008a;218: 629–638. pmid:18773219
- 6. Aldea D, Leon A, Bertrand S, Escriva H. Expression of Fox genes in the cephalochordate Branchiostoma lanceolatum. Front Ecol Evol 2015; http://dx.doi.org/10.3389/fevo.2015.00080
- 7. Fritzenwanker JH, Gerhart J, Freeman RM Jr, Lowe CJ. The Fox/Forkhead transcription factor family of the hemichordate Saccoglossus kowalevskii. Evodevo 2014;5: 17. pmid:24987514
- 8. Tu Q, Brown CT, Davidson EH, Oliveri P. Sea urchin Forkhead gene family: phylogeny and embryonic expression. Dev Biol 2006;300: 49–62. pmid:17081512
- 9. Shimeld SM, Boyle MJ, Brunet T, Luke GN, Seaver EC. Clustered Fox genes in lophotrochozoans and the evolution of the bilaterian Fox gene cluster. Dev Biol 2010a;340: 234–248. pmid:20096280
- 10. Fritzenwanker JH, Saina M, Technau U. Analysis of forkhead and snail expression reveals epithelial-mesenchymal transitions during embryonic and larval development of Nematostella vectensis. Dev Biol 2004;275: 389–402. pmid:15501226
- 11. Chevalier S, Martin A, Leclere L, Amiel A, Houliston E. Polarised expression of FoxB and FoxQ2 genes during development of the hydrozoan Clytia hemisphaerica. Dev Genes Evol 2006;216: 709–720. pmid:17021866
- 12. Adell T, Grebenjuk VA, Wiens M, Muller WEG. Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon. Dev Genes Evol 2003; 213: 421–434. pmid:12898249
- 13. Adell T, Muller WEG. Isolation and characterization of five Fox (Forkhead) genes from the sponge Suberites domuncula. Gene 2004;334: 35–46. pmid:15256253
- 14. Lee HH, Frasch M. Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns. Dev Dyn 2004;229: 357–366. pmid:14745961
- 15. Häcker U, Grossniklaus U, Gehring WJ, Jäckle H. Developmentally regulated Drosophila gene family encoding the fork head domain. Proc Natl Acad Sci U S A 1992;89: 8754–8758. pmid:1356269
- 16. Hope IA, Mounsey A, Bauer P, Aslam S. The forkhead gene family of Caenorhabditis elegans. Gene 2003;304: 43–55. pmid:12568714
- 17. Kittelmann S, Ulrich J, Posnien N, Bucher G. Changes in anterior head patterning underlie the evolution of long germ embryogenesis. Dev Biol 2013;374: 174–184. pmid:23201022
- 18. Janssen R, Jörgensen M, Lagebro L, Budd GE. Fate and nature of the onychophoran mouth-anus furrow and its contribution to the blastopore. Proc Biol Sci 2015;282: 1805.
- 19. Lin X, Yu N, Smagghe G. FoxO mediates the timing of pupation through regulating ecdysteroid biosynthesis in the red flour beetle, Tribolium castaneum. Gen Comp Endocrinol 2017;258: 149–156. pmid:28526479
- 20. Heingård M, Turetzek N, Prpic NM, Janssen R. FoxB, a new and highly conserved key factor in arthropod dorsal-ventral (DV) limb patterning. Evodevo 2019;10: 28. pmid:31728178
- 21. Hu Q, Zhu Z, Zhao D, Zeng B, Zheng S, Song Q, et al. Bombyx mori transcription factors FoxA and SAGE divergently regulate the expression of wing cuticle protein gene 4 during metamorphosis. J Biol Chem 2019;294: 632–643. pmid:30429222
- 22. Schacht MI, Schomburg C, Bucher G. six3 acts upstream of foxQ2 in labrum and neural development in the spider Parasteatoda tepidariorum. Dev Genes Evol 2020;230: 95–104. pmid:32040712
- 23. Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev 2000;14: 142–146. pmid:10702024
- 24. Mazet F, Yu JK, Liberles DA, Holland LZ, Shimeld SM. Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria. Gene 2003;316: 79–89. pmid:14563554
- 25. Heglind M, Cederberg A, Aquino J, Lucas G, Ernfors P, Enerbäck S. Lack of the central nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and body weight. Mol Cell Biol 2005;25: 5616–5625. pmid:15964817
- 26. Wotton KR, Shimeld SM. Comparative genomics of vertebrate fox cluster loci. BMC Genomics 2006;7: 271–278. pmid:17062144
- 27. Lin HY, Zhu CQ, Zhang HH, Shen ZC, Zhang CX, Ye YX. The Genetic Network of Forkhead Gene Family in Development of Brown Planthoppers. Biology (Basel) 2021;10: 867. pmid:34571744
- 28. Schomburg C, Janssen R, Prpic NM. Phylogenetic analysis of forkhead transcription factors in the Panarthropoda. Dev Genes Evol 2022;232: 39–48. pmid:35230523
- 29. Krause G. Die Eitypen der Insekten. Biol Zetralbl 1939;59: 495–536.
- 30. Liu PZ, Kaufman TC. Short and long germ segmentation: unanswered questions in the evolution of a developmental mode. Evol Dev 2005;7: 629–646. pmid:16336416
- 31. Giribet G, Edgecombe GD. Current Understanding of Ecdysozoa and its Internal Phylogenetic Relationships. Integr Comp Biol 2017;57: 455–466. pmid:28957525
- 32. Laumer CE, Fernández R, Lemer S, Combosch D, Kocot KM, Riesgo A, et al. Revisiting metazoan phylogeny with genomic sampling of all phyla. Proc Biol Sci 2019;286: 20190831. pmid:31288696
- 33. Grossmann D, Prpic NM. Egfr signaling regulates distal as well as medial fate in the embryonic leg of Tribolium castaneum. Dev Biol 2012;370: 264–272. pmid:22921411
- 34. Janssen R, Prpic NM, Damen WG. Gene expression suggests decoupled dorsal and ventral segmentation in the millipede Glomeris marginata (Myriapoda: Diplopoda). Dev Biol 2004;268: 89–104. pmid:15031107
- 35. Prpic NM, Schoppmeier M, Damen WG. Collection and fixation of spider embryos. CSH Protoc 2008. pmid:21356698
- 36. Hogvall M, Schönauer A, Budd GE, McGregor AP, Posnien N, Janssen R. Analysis of the Wnt gene repertoire in an onychophoran provides new insights into the evolution of segmentation. Evodevo 2014;5: 14. pmid:24708787
- 37. Strobl F, Stelzer EH. Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos. Development 2014;141: 2331–2338. pmid:24803590
- 38. Mittmann B, Wolff C. Embryonic development and staging of the cobweb spider Parasteatoda tepidariorum C. L. Koch, 1841 (syn.: Achaearanea tepidariorum; Araneomorphae; Theridiidae). Dev Genes Evol 2012;222: 189–216. pmid:22569930
- 39. Janssen R, Budd GE. Deciphering the onychophoran ’segmentation gene cascade’: Gene expression reveals limited involvement of pair rule gene orthologs in segmentation, but a highly conserved segment polarity gene network. Dev Biol 2013;382: 224–234. pmid:23880430
- 40. Janssen R, Andersson E, Betnér E, Bijl S, Fowler W, Höök L, et al. Embryonic expression patterns and phylogenetic analysis of panarthropod sox genes: insight into nervous system development, segmentation and gonadogenesis. BMC Evol Biol 2018:18: 88.
- 41. Schröder R, Eckert C, Wolff C, Tautz D. Conserved and divergent aspects of terminal patterning in the beetle Tribolium castaneum. Proc Natl Acad Sci U S A 2000;97: 6591–6596. pmid:10823887
- 42. Janssen R, Budd GE. Investigation of endoderm marker-genes during gastrulation and gut-development in the velvet worm Euperipatoides kanangrensis. Dev Biol 2017;427: 155–164. pmid:28465040
- 43. Akiyama-Oda Y, Oda H. Early patterning of the spider embryo: a cluster of mesenchymal cells at the cumulus produces Dpp signals received by germ disc epithelial cells. Development 2003;130: 1735–1747. pmid:12642480
- 44. Feitosa NM, Pechmann M, Schwager EE, Tobias-Santos V, McGregor AP, Damen WGM, et al. Molecular control of gut formation in the spider Parasteatoda tepidariorum. Genesis 2017;55. pmid:28432834
- 45. Heingård M, Janssen R. The forkhead box containing transcription factor FoxB is a potential component of dorsal-ventral body axis formation in the spider Parasteatoda tepidariorum. Dev Genes Evol 2020;230: 65–73. pmid:32034484
- 46. Economou AD, Telford MJ. Comparative gene expression in the heads of Drosophila melanogaster and Tribolium castaneum and the segmental affinity of the Drosophila hypopharyngeal lobes. Evol Dev 2009;11: 88–96. pmid:19196336
- 47. Janssen R, Budd GE, Damen WG. Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods. Dev Biol 2011a;357: 64–72. pmid:21658375
- 48. Snodgrass RE. Comparative studies on the jaws of mandibulate arthropods. Smithsonian Misc Coll 1950;116: 1–85.
- 49. Edgecombe GD. Morphological data, extant Myriapoda, and the myriapod stem-group. Contributions to Zoology 2004;73: 3.
- 50. Schomburg C, Turetzek N, Prpic NM. Candidate gene screen for potential interaction partners and regulatory targets of the Hox gene labial in the spider Parasteatoda tepidariorum. Dev Genes Evol 2020;230: 105–120. pmid:32036446
- 51. Janssen R. Segment polarity gene expression in a myriapod reveals conserved and diverged aspects of early head patterning in arthropods. Dev Genes Evol 2012;222: 299–309. pmid:22903234
- 52. Janssen R, Damen WG. Diverged and conserved aspects of heart formation in a spider. Evol Dev 2008;10: 155–165. pmid:18315809
- 53. Janssen R, Prpic NM, Damen WG. A review of the correlation of tergites, sternites, and leg pairs in diplopods. Front Zool 2006;3: 2. pmid:16451739
- 54. Janssen R. The embryonic expression pattern of a second, hitherto unrecognized, paralog of the pair-rule gene sloppy-paired in the beetle Tribolium castaneum. Dev Genes Evol 2020:230: 247–256. pmid:32430691
- 55. Choe CP, Brown SJ. Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum. Dev Biol 2007;302: 281–294. pmid:17054935
- 56. Janssen R, Budd GE, Prpic NM, Damen WGM. Expression of myriapod pair rule gene orthologs. EvoDevo 2011b;2: 5. pmid:21352542
- 57. Janssen R, Damen WG, Budd GE. Expression of pair rule gene orthologs in the blastoderm of a myriapod: evidence for pair rule-like mechanisms? BMC Dev Biol 2012;12: 15. pmid:22595029
- 58. Janssen R. Diplosegmentation in the pill millipede Glomeris marginata is the result of dorsal fusion. Evol Dev 2011;13: 477–487. pmid:23016908
- 59. Janssen R, Budd GE. Oscillating waves of Fox, Cyclin and CDK gene expression indicate unique spatiotemporal control of cell cycling during nervous system development in onychophorans. Arthropod Struct Dev 2021;62: 101042. pmid:33752095
- 60. Kitzmann P, Weißkopf M, Schacht MI, Bucher G. A key role for foxQ2 in anterior head and central brain patterning in insects. Development 2017;144: 2969–2981. pmid:28811313
- 61. Janssen R. Comparative analysis of gene expression patterns in the arthropod labrum and the onychophoran frontal appendages, and its implications for the arthropod head problem. Evodevo 2017;8: 1. pmid:28053697
- 62. Pascual-Carreras E, Herrera-Úbeda C, Rosselló M, Coronel-Córdoba P, Garcia-Fernàndez J, Saló E, et al. Analysis of Fox genes in Schmidtea mediterranea reveals new families and a conserved role of Smed-foxO in controlling cell death. Sci Rep 2021;11: 2947. pmid:33536473
- 63. Newton FG, zur Lage PI, Karak S, Moore DJ, Göpfert MC, Jarman AP. Forkhead transcription factor Fd3F cooperates with Rfx to regulate a gene expression program for mechanosensory cilia specialization. Dev Cell 2012;22: 1221–1233. pmid:22698283
- 64. Jürgens G, Weigel D. Terminal versus segmental development in the Drosophila embryo: the role of the homeotic gene fork head. Rouxs Arch Dev Biol 1988;197:345–354. pmid:28305430
- 65. Weigel D, Jürgens G, Küttner F, Seifert E, Jäckle H. The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell 1989;57: 645–658. pmid:2566386
- 66. Gaul U, Weigel D. Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo. Mech Dev 1990;33: 57–67. pmid:1982922
- 67. Mango SE, Lambie EJ, Kimble J. The pha-4 gene is required to generate the pharyngeal primordium of Caenorhabditis elegans. Development 1994;120: 3019–3031. pmid:7607089
- 68. Kalb JM, Lau KK, Goszczynski B, Fukushige T, Moons D, Okkema PG, et al. pha-4 is Ce-fkh-1, a fork head/HNF-3alpha, beta, gamma homolog that functions in organogenesis of the C. elegans pharynx. Development 1998;125: 2171–2180. pmid:9584117
- 69. Martin-Duran JM, Janssen R, Wennberg S, Budd GE, Hejnol A. Deuterostomic development in the protostome Priapulus caudatus. Curr Biol 2012;22: 2161–2166. pmid:23103190
- 70. Boyle MJ, Seaver EC. Developmental expression of foxA and gata genes during gut formation in the polychaete annelid, Capitella sp I. Evol Dev 2008;10: 89–105. pmid:18184360
- 71. Boyle MJ, Seaver EC. Expression of FoxA and GATA transcription factors correlates with regionalized gut development in two lophotrochozoan marine worms: Chaetopterus (Annelida) and Themiste lageniformis (Sipuncula). Evodevo 2010;1: 2. pmid:20849645
- 72. Martin-Duran JM, Amaya E, Romero R. Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa. Dev Biol 2010;340: 145–158. pmid:20100474
- 73. Kwak HJ, Ryu KB, Medina Jiménez BI, Park SC, Cho SJ. Temporal and spatial expression of the Fox gene family in the Leech Helobdella austinensis. J Exp Zool B Mol Dev Evol 2018;330: 341–350. pmid:30280505
- 74. Kostyuchenko RP, Kozin VV, Filippova NA, Sorokina EV. FoxA expression pattern in two polychaete species, Alitta virens and Platynereis dumerilii: Examination of the conserved key regulator of the gut development from cleavage through larval life, postlarval growth, and regeneration. Dev Dyn 2019;248: 728–743. pmid:30566266
- 75. Taguchi S, Tagawa K, Humphreys T, Nishino A, Satoh N, Harada Y. Characterization of a hemichordate fork head/HNF-3 gene expression. Dev Genes Evol 2000;210: 11–17. pmid:10603081
- 76. Oliveri P, Walton KD, Davidson EH, McClay DR. Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo. Development 2006;133: 4173–4781. pmid:17038513
- 77. Martinez DE, Dirksen M-L, Bode PM, JamrichM, Steele RE, Bode HR. Budhead, a Fork Head/HNF-3 homologue, is expressed during axis formation and head specification in Hydra. Dev Biol 1997;192: 523–536. pmid:9441686
- 78. Miller LM, Waring DA, Kim SK. Mosaic analysis using a ncl-1 (+) extrachromosomal array reveals that lin-31 acts in the Pn.p cells during Caenorhabditis elegans vulval development. Genetics 1996;143: 1181–1191. pmid:8807292
- 79. Tan PB, Lackner MR, Kim SK. MAP kinase signaling specificity mediated by the LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction. Cell 1998;93: 569–580. pmid:9604932
- 80. Luke NH, Killian CE, Livingston BT. Spfkh1 encodes a transcription factor implicated in gut formation during sea urchin development. Dev Growth Differ 1997;39: 285–294. pmid:9227895
- 81. Minokawa T, Rast JP, Arenas-Mena C, Franco CB, Davidson EH. Expression patterns of four different regulatory genes that function during sea urchin development. Gene Expr Patterns 2004;4: 449–456. pmid:15183312
- 82. Czarkwiani A, Dylus DV, Oliveri P. Expression of skeletogenic genes during arm regeneration in the brittle star Amphiura filiformis. Gene Expr Patterns 2013;13: 464–472. pmid:24051028
- 83. Odenthal J, Nüsslein-Volhard C. fork head domain genes in zebrafish. Dev Genes Evol 1998;208: 245–258. pmid:9683740
- 84. Gamse JT, Sive H. Early anteroposterior division of the presumptive neurectoderm in Xenopus. Mech Dev 2001;104: 21–36. pmid:11404077
- 85. Arendt D, Nübler-Jung K. Dorsal or ventral: similarities in fate maps and gastrulation patterns in annelids, arthropods and chordates. Mech Dev 1997;61: 7–21. pmid:9076674
- 86. Häcker U, Kaufmann E, Hartmann C, Jürgens G, Knöchel W, Jäckle H. The Drosophila fork head domain protein crocodile is required for the establishment of head structures. EMBO J 1995;14: 5306–5317. pmid:7489720
- 87. Birkan M, Schaeper ND, Chipman AD. Early patterning and blastodermal fate map of the head in the milkweed bug Oncopeltus fasciatus. Evol Dev 2011;13: 436–447. pmid:23016905
- 88. Passamaneck YJ, Hejnol A, Martindale MQ. Mesodermal gene expression during the embryonic and larval development of the articulate brachiopod Terebratalia transversa. Evodevo 2015;6: 10. pmid:25897375
- 89. Wotton KR, Mazet F, Shimeld SM. Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish Scyliorhinus canicula defines ancient and derived roles for Fox genes in vertebrate development. Dev Dyn 2008;237: 1590–1603. pmid:18498098
- 90. Magie CR, Pang K, Martindale MQ. Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis. Dev Genes Evol 2005;215: 618–30. pmid:16193320
- 91. Martindale MQ, Pang K, Finnerty JR (2004) Investigating the origins of triploblasty: ‘mesodermal’ gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa). Development 2004;131: 2463–2474. pmid:15128674
- 92. Lacin H, Rusch J, Yeh RT, Fujioka M, Wilson BA, Zhu Y, et al. Genome-wide identification of Drosophila Hb9 targets reveals a pivotal role in directing the transcriptome within eight neuronal lineages, including activation of nitric oxide synthase and Fd59a/Fox-D. Dev Biol 2014;388: 117–133. pmid:24512689
- 93. Nash B, Colavita A, Zheng H, Roy PJ, Culotti JG. The forkhead transcription factor UNC-130 is required for the graded spatial expression of the UNC-129 TGF-beta guidance factor in C. elegans. Genes Dev 2000;14: 2486–2500. pmid:11018016
- 94. Sarafi-Reinach TR, Sengupta P. The forkhead domain gene unc-130 generates chemosensory neuron diversity in C. elegans. Genes Dev 2000;14: 2472–2485. pmid:11018015
- 95. Kersey RK, Brodigan TM, Fukushige T, Krause MW. Regulation of UNC-130/FOXD-mediated mesodermal patterning in C. elegans. Dev Biol 2016;416: 300–311. pmid:27341757
- 96. Shen Q, Toulabi LB, Shi H, Nicklow EE, Liu J. The forkhead transcription factor UNC-130/FOXD integrates both BMP and Notch signaling to regulate dorsoventral patterning of the C. elegans postembryonic mesoderm. Dev Biol 2018;433: 75–83. pmid:29155044
- 97. Lauri A, Brunet T, Handberg-Thorsager M, Fischer AHL, Simakov O, Steinmetz PRH, et al. Development of the annelid axochord: insights into notochord evolution. Science 2014;345: 1365–1368. pmid:25214631
- 98. Yankura KA, Martik ML, Jennings CK, Hinman VF 2010. Uncoupling of complex regulatory patterning during evolution of larval development in echinoderms. BMC Biol 8:143. pmid:21118544
- 99. Imai KS, Satoh N, Satou Y. An essential role of a FoxD gene in notochord induction in Ciona embryos. Development 2002;129: 3441–3453. pmid:12091314
- 100. Hudson C, Sirour C, Yasuo H. Co-expression of Foxa.a, Foxd and Fgf9/16/20 defines a transient mesendoderm regulatory state in ascidian embryos. Elife 2016; e14692. pmid:27351101
- 101. Tokuhiro SI, Tokuoka M, Kobayashi K, Kubo A, Oda-Ishii I, Satou Y. Differential gene expression along the animal-vegetal axis in the ascidian embryo is maintained by a dual functional protein Foxd. PLoS Genet 2017;13: e1006741. pmid:28520732
- 102. Yu JK, Holland ND, Holland LZ. An amphioxus winged helix/forkhead gene, AmphiFoxD: insights into vertebrate neural crest evolution. Dev Dyn 2002;225: 289–297. pmid:12412011
- 103. Zaffran S, Küchler A, Lee HH, Frasch M. biniou (FoxF), a central component in a regulatory network controlling visceral mesoderm development and midgut morphogenesis in Drosophila. Genes Dev 2001;15: 2900–2915. pmid:11691840
- 104. Perez Sanchez C, Casas-Tinto S, Sanchez L, Rey-Campos J, Granadino B. Dm Fox F, a novel Drosophila fork head factor expressed in visceral mesoderm. Mech Dev 2002;111: 163–166. pmid:11804790
- 105. Jakobsen JS, Braun M, Astorga J, Gustafson EH, Sandmann T, Karzynski M, et al. Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network. Genes Dev 2007;21: 2448–2460. pmid:17908931
- 106. Popichenko D, Sellin J, Bartkuhn M, Paululat A. Hand is a direct target of the forkhead transcription factor Biniou during Drosophila visceral mesoderm differentiation. BMC Dev Biol 2007;7: 49. pmid:17511863
- 107. Carlsson P, Mahlapuu M. Forkhead transcription factors: key players in development and metabolism. Dev Biol 2002;250: 1–23. pmid:12297093
- 108. Amin NM, Shi H, Liu J. The FoxF/FoxC factor LET-381 directly regulates both cell fate specification and cell differentiation in C. elegans mesoderm development. Development 2010;137: 1451–1460. pmid:20335356
- 109. Scimone ML, Wurtzel O, Malecek K, Fincher CT, Oderberg IM, Kravarik KM, et al. foxF-1 Controls Specification of Non-body Wall Muscle and Phagocytic Cells in Planarians. Curr Biol 2018;28: 3787–3801. pmid:30471994
- 110. Ormestad M, Astorga J, Landgren H, Wang T, Johansson BR, Miura N, et al. Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production. Development 2006;133: 833–843. pmid:16439479
- 111. Beh J, Shi W, Levine M, Davidson B, Christiaen L. FoxF is essential for FGF-induced migration of heart progenitor cells in the ascidian Ciona intestinalis. Development 2007;134: 3297–3305. pmid:17720694
- 112. Andrikou C, Iovene E, Rizzo F, Oliveri P, Arnone MI. Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors. Evodevo 2013;4: 33. pmid:24295205
- 113. Wijesena N, Simmons DK, Martindale MQ. Antagonistic BMP-cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm. Proc Natl Acad Sci U S A 2017;114: E5608–E5615. pmid:28652368
- 114. Steinmetz PRH, Aman A, Kraus JEM, Technau U. Gut-like ectodermal tissue in a sea anemone challenges germ layer homology. Nat Ecol Evol 2017;1: 1535–1542. pmid:29185520
- 115. Grossniklaus U, Pearson RK, Gehring WJ. The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. Genes Dev 1992;6: 1030–1051. pmid:1317319
- 116. Cadigan KM, Grossniklaus U, Gehring WJ. Localized expression of sloppy paired protein maintains the polarity of Drosophila parasegments. Genes Dev 1994a;8: 899–913. pmid:7926775
- 117. Cadigan KM, Grossniklaus U, Gehring WJ. Functional redundancy: the respective roles of the two sloppy paired genes in Drosophila segmentation. Proc Natl Acad Sci U S A 1994b;91: 6324–6328. pmid:8022780
- 118. Damen WG, Janssen R, Prpic NM. Pair rule gene orthologs in spider segmentation. Evol Dev 2005;7: 618–628. pmid:16336415
- 119. Green J, Akam M. Evolution of the pair rule gene network: Insights from a centipede. Dev Biol 2013;382: 235–245. pmid:23810931
- 120. Reding K, Chen M, Lu Y, Jarvela AMC, Pick L. Shifting roles of Drosophila pair-rule gene orthologs: segmental expression and function in the milkweed bug Oncopeltus fasciatus. Development 2019;146: dev181453. pmid:31444220
- 121. Grossniklaus U, Cadigan KM, Gehring WJ. Three maternal coordinate systems cooperate in the patterning of the Drosophila head. Development 1994; 120: 3155–3171. pmid:7720559
- 122. Mukhopadhyay S, Lu Y, Qin H, Lanjuin A, Shaham S, Sengupta P. Distinct IFT mechanisms contribute to the generation of ciliary structural diversity in C. elegans. EMBO J 2007;26: 2966–2980. pmid:17510633
- 123. Santagata S, Resh C, Hejnol A, Martindale MQ, Passamaneck YJ. Development of the larval anterior neurogenic domains of Terebratalia transversa (Brachiopoda) provides insights into the diversification of larval apical organs and the spiralian nervous system. Evodevo 2012;3: 3. pmid:22273002
- 124. Kerbl A, Martín-Durán JM, Worsaae K, Hejnol A. Molecular regionalization in the compact brain of the meiofaunal annelid Dinophilus gyrociliatus (Dinophilidae). Evodevo 2016;7: 20. pmid:27583125
- 125. Koinuma S, Umesono Y, Watanabe K, Agata K. The expression of planarian brain factor homologs, DjFoxG and DjFoxD. Gene Expr Patterns 2003;3: 21–27. pmid:12609597
- 126. Yankura KA, Koechlein CS, Cryan AF, Cheatle A, Hinman VF. Gene regulatory network for neurogenesis in a sea star embryo connects broad neural specification and localized patterning. Proc Natl Acad Sci U S A 2013;110: 8591–8596. pmid:23650356
- 127. Toresson H, Martinez-Barbera JP, Bardsley A, Caubit X, Krauss S. Conservation of BF-1 expression in amphioxus and zebrafish suggests evolutionary ancestry of anterior cell types that contribute to the vertebrate telencephalon. Dev Genes 1998;208: 431–439.
- 128. Roth M, Bonev B, Lindsay J, Lea R, Panagiotaki N, Houart C, et al. FoxG1 and TLE2 act cooperatively to regulate ventral telencephalon formation. Development 2010;137: 1553–1562. pmid:20356955
- 129. Yoshida K, Saiga H. Left-right asymmetric expression of Pitx is regulated by the asymmetric Nodal signaling through an intronic enhancer in Ciona intestinalis. Dev Genes Evol 2008;218: 353–360. pmid:18546017
- 130. Vieillard J, Duteyrat JL, Cortier E, Durand B. Imaging cilia in Drosophila melanogaster. Methods Cell Biol. 2015;127:279–302. pmid:25837397
- 131. Yu X, Ng CP, Habacher H, Roy S. Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat Genet 2008b;40: 1445–1453. pmid:19011630
- 132. Vij S, Rink JC, Ho HK, Babu D, Eitel M, Narasimhan V, et al. Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program. PLoS Genet. 2012;8(11):e1003019. pmid:23144623
- 133. Bonnafe E, Touka M, AitLounis A, Baas D, Barras E, Ucla C, et al. The transcription factor RFX3 directs nodal cilium development and left-right asymmetry specification. Mol Cell Biol. 2004 May;24(10):4417–27. pmid:15121860
- 134. Laurençon A, Dubruille R, Efimenko E, Grenier G, Bissett R, Cortier E, et al. Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species. Genome Biol. 2007;8(9):R195. pmid:17875208
- 135. Chen N, Mah A, Blacque OE, Chu J, Phgora K, Bakhoum MW, et al. Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics. Genome Biol. 2006;7(12):R126. pmid:17187676
- 136. Tomancak P, Beaton A, Weiszmann R, Kwan E, Shu S, Lewis SE, et al. Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol 2002;3: 0088 pmid:12537577
- 137. Tomancak P, Berman BP, Beaton A, Weiszmann R, Kwan E, Hartenstein V, et al. Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol 2007; 8: R145. pmid:17645804
- 138. Marlow H, Tosches MA, Tomer R, Steinmetz PR, Lauri A, Larsson T, et al. Larval body patterning and apical organs are conserved in animal evolution. BMC Biol 2014;12:7. pmid:24476105
- 139. Gomperts BN, Gong-Cooper X, Hackett BP. Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells. J Cell Sci 2004;117: 1329–1337. pmid:14996907
- 140. Stubbs JL, Oishi I, Izpisúa Belmonte JC, Kintner C. The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. Nat Genet 2008;40: 1454–1460. pmid:19011629
- 141. Sinigaglia C, Busengdal H, Leclere L, Technau U, Rentzsch F. The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian. PLoS Biol 2013;11: e1001488 pmid:23483856
- 142. Larroux C, Fahey B, Liubicich D, Hinman VF, Gauthier M, Gongora M, et al. Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity. Evol Dev 2006;8: 150–173. pmid:16509894
- 143. King N, Westbrook MJ, Young SL, Kuo A, Abedin M, Chapman J, et al. The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans. Nature 2008;451: 783–788. pmid:18273011
- 144. Choi VM, Harland RM, Khokha MK. Developmental expression of FoxJ1.2, FoxJ2, and FoxQ1 in Xenopus tropicalis. Gene Expr Patterns 2006;6: 443–447. pmid:16461016
- 145. Miao H, Miao CX, Li N, Han J. FOXJ2 controls meiosis during spermatogenesis in male mice. Mol Reprod Dev 2016;83: 684–691. pmid:27316861
- 146. Zhang Z, Meng G, Wang L, Ma Y, Guan Z. The prognostic role and reduced expression of FOXJ2 in human hepatocellular carcinoma. Mol Med Rep 2016;14: 254–262. pmid:27177166
- 147. Landgren H, Carlsson P. FoxJ3, a novel mammalian forkhead gene expressed in neuroectoderm, neural crest, and myotome. Dev Dyn 2004;231: 396–401. pmid:15366017
- 148. Tomczyk S, Buzgariu W, Perruchoud C, Fisher K, Austad S, Galliot B. Loss of Neurogenesis in Aging Hydra. Dev Neurobiol 2019;79: 479–496. pmid:30912256
- 149. Casas-Tinto S, Gomez-Velazquez M, Granadino B, Fernandez-Funez P. FoxK mediates TGF-beta signalling during midgut differentiation in flies. J Cell Biol 2008;183: 1049–1060. pmid:19075113
- 150. Liu Y, Ding W, Ge H, et al. FOXK transcription factors: Regulation and critical role in cancer. Cancer Lett 2019;458: 1–12. pmid:31132431
- 151. Hanlon CD, Andrew DJ. Drosophila FoxL1 non-autonomously coordinates organ placement during embryonic development. Dev Biol 2016;419: 273–284. pmid:27618755
- 152. Kato Y, Fukamachi H, Takano-Maruyama M, Aoe T, Murahashi Y, Horie S, et al. Reduction of SNAP25 in acid secretion defect of Foxl1-/- gastric parietal cells. Biochem Biophys Res Commun 2004;320: 766–772. pmid:15240114
- 153. Katz JP, Perreault N, Goldstein BG, Chao HH, Ferraris RP, Kaestner KH. Foxl1 null mice have abnormal intestinal epithelia, postnatal growth retardation, and defective intestinal glucose uptake. Am J Physiol Gastrointest Liver Physiol 2004;287: G856–G864. pmid:15155178
- 154. Ye YX, Pan PL, Xu JY, Shen ZF, Kang D, Lu JB, et al. Forkhead box transcription factor L2 activates Fcp3C to regulate insect chorion formation. Open Biol 2017;7: 170061. pmid:28615473
- 155. Cocquet J, Pailhoux E, Jaubert F, Servel N, Xia X, Pannetier M, et al. Evolution and expression of Foxl2. J Med Genet 2002;39: 916–921. pmid:12471206
- 156. Naimi A, Martinez AS, Specq ML, Diss B, Mathieu M, Sourdaine P. Molecular cloning and gene expression of Cg-Foxl2 during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas. Comp Biochem Physiol B Biochem Mol Biol 2009;154: 134–142. pmid:19481171
- 157. Cocquet J, Pannetier M, Fellousa M, Veitiaa RA. Sense and antisense Foxl2 transcripts in mouse. Genomics 2005;85: 531–541. pmid:15820304
- 158. Santerre C, Sourdaine P, Martinez AS. Expression of a natural antisens transcript of Cg-Foxl2 during the gonadic differentiation of the oyster Crassostrea gigas: first demonstration in the gonads of a lophotrochozoa species. Sex Dev 2012;6: 210–221. pmid:22572405
- 159. Cheah PY, Chia W, Yang X. Jumeaux, a novel Drosophila winged-helix family protein, is required for generating asymmetric sibling neuronal cell fates. Development 2000;127: 3325–3335. pmid:10887088
- 160. Sugimura I, Adachi-Yamada T, Nishi Y, Nishida Y. A Drosophila Winged-helix nude (Whn)-like transcription factor with essential functions throughout development. Dev Growth Differ 2000;42: 237–248. pmid:10910130
- 161. Ahmad SM, Tansey TR, Busser BW, Nolte MT, Jeffries N, Gisselbrecht SS, et al. Two forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway. Dev Cell 2012;23: 97–111. pmid:22814603
- 162. Zhu X, Ahmad SM, Aboukhalil A, Busser BW, Kim Y, Tansey TR, et al. Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors. Development 2012;139: 1457–1466. pmid:22378636
- 163. Golson ML, Kaestner KH. Fox transcription factors: from development to disease. Development 2016;143: 4558–4570. pmid:27965437
- 164. Liao GB, Li XZ, Zeng S, et al. Regulation of the master regulator FOXM1 in cancer. Cell Commun Signal 2018;16: 57. pmid:30208972
- 165. Prowse DM, Lee D, Weiner L, Jiang N, Magro CM, Baden HP, et al. Ectopic expression of the nude gene induces hyperproliferation and defects in differentiation: Implications for the self-renewal of cutaneous epithelia. Dev Biol 1999;212: 54–67. pmid:10419685
- 166. Huot G, Vernier M, Bourdeau V, Doucet L, Saint-Germain E, Gaumont-Leclerc MF, et al. CHES1/FOXN3 regulates cell proliferation by repressing PIM2 and protein biosynthesis. Mol Biol Cell 2014;25: 554–565. pmid:24403608
- 167. Sun J, Li H, Huo Q, Cui M, Ge C, Zhao F, et al. The transcription factor FOXN3 inhibits cell proliferation by downregulating E2F5 expression in hepatocellular carcinoma cells. Oncotarget 2016;7: 43534–43545. pmid:27259277
- 168. Riddle DL, Swanson MM, Albert PS. Interacting genes in nematode dauer larva formation. Nature 1981;290: 268–271. pmid:7219552
- 169. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. A C. elegans mutant that lives twice as long as wild type. Nature 1993;366: 461–464. pmid:8247153
- 170. Kim SY, Webb AE. Neuronal functions of FOXO/DAF-16. Nutr Healthy Aging 2017;4: 113–126. pmid:28447066
- 171. Koyama T, Rodrigues MA, Athanasiadis A, Shingleton AW, Mirth CK. Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis. Elife 2014;3: e03091. pmid:25421296
- 172. Zeng B, Huang Y, Xu J, Shiotsuki T, Bai H, Palli SR, et al. The FOXO transcription factor controls insect growth and development by regulating juvenile hormone degradation in the silkworm, Bombyx mori. J Biol 2017;292: 11659–11669. pmid:28490635
- 173. Lu Z, Meng Z, Wen M, et al. Overexpression of BmFoxO inhibited larval growth and promoted glucose synthesis and lipolysis in silkworm. Mol Genet Genomics. 2019;294: 1375–1383. pmid:31214765
- 174. Arden KC. FOXO animal models reveal a variety of diverse roles for FOXO transcription factors. Oncogene 2008;27: 2345–2350. pmid:18391976
- 175. Mortzfeld BM, Taubenheim J, Fraune S, Klimovich AV, Bosch TCG. Stem Cell Transcription Factor FoxO Controls Microbiome Resilience in Hydra. Front Microbiol 2018;9: 629. pmid:29666616
- 176. Bosch TCG. Hydra as Model to Determine the Role of FOXO in Longevity. Methods Mol Biol 2019;1890: 231–238. pmid:30414158
- 177. DasGupta S, Ferreira CH, Miesenböck G. FoxP influences the speed and accuracy of a perceptual decision in Drosophila. Science 2014;344: 901–904. pmid:24855268
- 178. Lawton KJ, Wassmer TL, Deitcher DL. Conserved role of Drosophila melanogaster FoxP in motor coordination and courtship song. Behav Brain Res 2014;268: 213–221. pmid:24747661
- 179. Castells-Nobau A, Eidhof I, Fenckova M, Brenman-Suttner DB, Scheffer-de Gooyert JM, Christine S, et al. Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila. PLoS One 2019;14: e0211652. pmid:30753188
- 180. Kiya T, Itoh Y, Kubo T. Expression analysis of the FoxP homologue in the brain of the honeybee, Apis mellifera. Insect Mol Biol 2008;17: 53–60. pmid:18237284
- 181. Schatton A, Mendoza E, Grube K, Scharff C. FoxP in bees: A comparative study on the developmental and adult expression pattern in three bee species considering isoforms and circuitry. J Comp Neurol 2018;526: 1589–1610. pmid:29536541
- 182. Ferland RJ, Cherry TJ, Preware PO, Morrisey EE, Walsh CA. Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. J Comp Neurol 2003;460: 266–279. pmid:12687690
- 183. Bonkowsky JL, Chien CB. Molecular cloning and developmental expression of foxP2 in zebrafish. Dev Dyn 2005;234: 740–746. pmid:16028276
- 184. Rousso DL, Pearson CA, Gaber ZB, Miquelajauregui A, Li S, Portera-Cailliau C, et al. Foxp-mediated suppression of N-cadherin regulates neuroepithelial character and progenitor maintenance in the CNS. Neuron 2012;74: 314–330. pmid:22542185
- 185. Shimeld SM, Degnan B, Luke GN. Evolutionary genomics of the Fox genes: Origin of gene families and the ancestry of gene clusters. Genomics 2010b;95: 256–260. pmid:19679177
- 186. Ogasawara M, Satou Y. Expression of FoxE and FoxQ genes in the endostyle of Ciona intestinalis. Dev Genes Evol 2003;213: 416–419. pmid:12802588
- 187. Martin-Duran JM, Vellutini BC, Hejnol A. Evolution and development of the adelphophagic, intracapsular Schmidt’s larva of the nemertean Lineus ruber. Evodevo 2015;6: 28. pmid:26417429
- 188. Hunnekuhl VS, Akam M. An anterior medial cell population with an apical-organ-like transcriptional profile that pioneers the central nervous system in the centipede Strigamia maritima. Dev Biol 2014;396: 136–149. pmid:25263198
- 189. He B, Buescher M, Farnworth MS, et al. An ancestral apical brain region contributes to the central complex under the control of foxQ2 in the beetle Tribolium. Elife 2019;8: e49065. pmid:31625505
- 190. Steinmetz PR, Urbach R, Posnien N, Eriksson J, Kostyuchenko RP, Brena C, et al. Six3 demarcates the anterior-most developing brain region in bilaterian animals. Evodevo 2010;1: 14. pmid:21190549
- 191. Posnien N, Koniszewski ND, Hein HJ, Bucher G. Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development. PLoS Genet 2011;7: e1002416. pmid:22216011
- 192. Eriksson BJ, Samadi L, Schmid A. The expression pattern of the genes engrailed, pax6, otd and six3 with special respect to head and eye development in Euperipatoides kanangrensis Reid 1996 (Onychophora: Peripatopsidae). Dev Genes Evol 2013;223: 237–246. pmid:23625086
- 193. Lowe CJ, Wu M, Salic A, Evans L, Lander E, Stange-Thomann N, et al. Anteroposterior patterning in hemichordates and the origins of the chordate nervous system. Cell 2003;113: 853–865. pmid:12837244
- 194. Range RC, Wei Z. An anterior signaling center patterns and sizes the anterior neuroectoderm of the sea urchin embryo. Development 2016;143: 1523–1533. pmid:26952978
- 195. Redl E, Scherholz M, Wollesen T, Todt C, Wanninger A. Expression of six3 and otx in Solenogastres (Mollusca) supports an ancestral role in bilaterian anterior-posterior axis patterning. Evol Dev 2018;20: 17–28. pmid:29243871
- 196. Cachero S, Simpson TI, Zur Lage PI, Ma L, Newton FG, Holohan EE, et al. The gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons. PLoS Biol 2011;9: e1000568. pmid:21283833
- 197. Rouyer F, Rachidi M, Pikielny C, Rosbash M. A new gene encoding a putative transcription factor regulated by the Drosophila circadian clock. EMBO J 1997;16: 3944–3954. pmid:9233804
- 198. Nie H, Geng H, Lin Y, et al. Genome-Wide Identification and Characterization of Fox Genes in the Honeybee, Apis cerana, and Comparative Analysis with Other Bee Fox Genes. Int J Genomics 2018; 5702061. pmid:29850474