Extensive loss of Wnt genes in Tardigrada

Abstract

Background: Wnt genes code for ligands that activate signaling pathways during development in Metazoa. Through the canonical Wnt (cWnt) signaling pathway, these genes regulate important processes in bilaterian development, such as establishing the anteroposterior axis and posterior growth. In Arthropoda, Wnt ligands also regulate segment polarity, and outgrowth and patterning of developing appendages. Arthropods are part of a lineage called Panarthropoda that includes Onychophora and Tardigrada. Previous studies revealed potential roles of Wnt genes in regulating posterior growth, segment polarity, and growth and patterning of legs in Onychophora. Unlike most other panarthropods, tardigrades lack posterior growth, but retain segmentation and appendages. Here, we investigated Wnt genes in tardigrades to gain insight into potential roles that these genes play during development of the highly compact and miniaturized tardigrade body plan.

Results: We analyzed published genomes for two representatives of Tardigrada, Hypsibius exemplaris and Ramazzottius varieornatus. We identified single orthologs of Wnt4, Wnt5, Wnt9, Wnt11, and WntA, as well as two Wnt16 paralogs in both tardigrade genomes. We only found a Wnt2 ortholog in H. exemplaris. We could not identify orthologs of Wnt1, Wnt6, Wnt7, Wnt8, or Wnt10. We identified most other components of cWnt signaling in both tardigrade genomes. However, we were unable to identify an ortholog of arrow/Lrp5/6, a gene that codes for a Frizzled co-receptor of Wnt ligands. Additionally, we found that some other animals that have lost several Wnt genes and are secondarily miniaturized, like tardigrades, are also missing an ortholog of arrow/Lrp5/6. We analyzed the embryonic expression patterns of Wnt genes in H. exemplaris during developmental stages that span the establishment of the AP axis through segmentation and leg development. We detected expression of all Wnt genes in H. exemplaris besides one of the Wnt16 paralogs. During embryo elongation, expression of several Wnt genes was restricted to the posterior pole or a region between the anterior and posterior poles. Wnt genes were expressed in distinct patterns during segmentation and development of legs in H. exemplaris, rather than in broadly overlapping patterns.

Conclusions: Our results indicate that Wnt signaling has been highly modified in Tardigrada. While most components of cWnt signaling are conserved in tardigrades, we conclude that tardigrades have lost Wnt1, Wnt6, Wnt7, Wnt8, and Wnt10, along with arrow/Lrp5/6. Our expression data may indicate a conserved role of Wnt genes in specifying posterior identities during establishment of the AP axis. However, the loss of several Wnt genes and the distinct expression patterns of Wnt genes during segmentation and leg development may indicate that combinatorial interactions among Wnt genes are less important during tardigrade development compared to many other animals. Based on our results, and comparisons to previous studies, we speculate that the loss of several Wnt genes in Tardigrada may be related to a reduced number of cells and simplified development that accompanied miniaturization and anatomical simplification in this lineage.

Keywords: Miniaturization; Panarthropoda; Posterior growth; Tardigrada; Wnt signaling.

Fig. 1

Majority rule consensus tree of Wnt ligands. For the sake of space, taxon abbreviations are used, and branch lengths are not diagrammed. See Additional file 1: Fig. S1 for a version of this tree that includes branch length information. For simplicity, only branch support values relevant to determining the identity of the candidate tardigrade Wnt ligands are shown. Tardigrade sequences are in colored boxes. Bootstrap supports are shown as percentages out of 500 replicates. Species abbreviations: Ap, Acyrthosiphon pisum; Cs, Cupiennius salei; Dp, Daphnia pulex; Dm, Drosophila melanogaster; Ek, Euperipatoides kanangrensis; Gm, Glomeris marginata; He, Hypsibius exemplaris; Hs, Homo sapiens; Is, Ixodes scapularis; Pd, Platynereis dumerilii; Pt, Parasteatoda tepidariorum; Rv, Ramazzottius varieornatus; Tc, Tribolium castaneum

Fig. 2

Majority rule consensus tree of Frizzled receptors. Tardigrade sequences are in colored boxes. Bootstrap supports are shown as percentages out of 500 replicates. For simplicity, only branch support values relevant to determining the identity of the candidate tardigrade Frizzled receptors are shown. Species abbreviations: Ct, Capitella teleta; Cg, Crassostrea gigas; Dp, Daphnia pulex; Ek, Euperipatoides kanangrensis; Gm, Glomeris marginata; He, Hypsibius exemplaris; Is, Ixodes scapularis; Mm, Mus musculus; Pt, Parasteatoda tepidariorum; Rv, Ramazzottius varieornatus; Tc, Tribolium castaneum; Xl, Xenopus laevis; Zn, Zootermopsis nevadensis

Fig. 3

Conserved domains in LRP sequences. Blue boxes represent low-density lipoprotein receptor repeat class B domains. Gold boxes represent calcium-binding EGF-like domains. Green boxes represent low-density lipoprotein receptor class A domains. a Pattern of conserved domains in Arrow/LRP5/6 orthologs. b General pattern of conserved domains in Arrow/LRP5/6 orthologs. c Pattern of conserved domains in the best matches to Arrow/LRP5/6 in species that have lost several Wnt ligands. The lower case letter after the H. miamia sequence does not indicate specific orthology. The specific orthology of this sequence is unclear based on our analyses. The S. mediterranea sequence represents a highly derived Arrow/LRP5/6 ortholog based on functional analyses [98]

Fig. 4

Majority rule consensus tree of LRP sequences. Tardigrade sequences are in colored boxes. Bootstrap supports are shown as percentages out of 500 bootstrap replicates. For simplicity, only branch support values relevant to determining the identity of the candidate tardigrade LRP sequences are shown. Lower case letters after taxon names indicate unclear orthology. The orthology of the S. mediterranea LRP5/6 sequence is based on functional analyses [98]. Species abbreviations: Ad, Adineta vaga; Ce, Caenorhabditis elegans; Dg, Dimorphilus gyrociliatus; Dm, Drosophila melanogaster; Ek, Euperipatoides kanangrensis; He, Hypsibius exemplaris; Hm, Hofstenia miamia; Mm, Mus musculus; Ob, Octopus bimaculoides; Rv, Ramazzottius varieornatus; Sm, Schmidtea mediterranea; Tc, Tribolium castaneum

Fig. 5

Elongation, segmentation, and leg development stages in H. exemplaris. Panels to the left show DAPI stained embryos. Models are provided to the right of each data panel. The key for the color-coding in models is provided at the bottom of the figure. a Early elongation (stage 11). b Late elongation (stage 11). a, b Arrowhead points to internalized cells that connect to the external ectoderm. c, d Stage 12. e, f Stage 13. e–e’’ Views from more lateral to more medial. e, f Dashed lines in the model denote the position of ectodermal furrows. g, h Stage 14. Anterior is towards the top in all panels. All panels show a lateral view of embryos facing right except for d, f, and h, which show bilateral views. All panels show internal anatomy, except for e and g, which show external features. ant anterior; ep1–ep4 endomesodermal pouch 1–endomesodermal pouch 4; fg foregut; l1–l4 leg 1–leg 4; pos posterior, t1–t4 trunk segment 1–trunk segment 4

Fig. 6

In situ hybridization results for Wnt genes in stage 11 H. exemplaris embryos. Green color represents gene expression. Nuclei are labeled with DAPI (blue). Images that share the same letter represent data from the same embryo. Arrowheads point to internal cells that connect to the ectoderm at the posterior end. Anterior is towards the top. a Six3 expression in early elongation stage embryos. b Six3 expression in late elongation stage embryos. c Wnt2 expression. d Wnt 4 expression. e, f Wnt5 expression in early elongation stage embryos. g Wnt5 expression in late elongation stage embryos. h Wnt11 expression. i Wnt16B expression. j WntA expression. f, g’, h’’’, j’ Bilateral view showing internal anatomy. h’’ Bilateral view of outer ectoderm. All other panels show lateral views of embryos that are facing right. a’–d’, h’, i’ show internal anatomy of laterally viewed embryos

Fig. 7

In situ hybridization results for Wnt genes in stage 12 H. exemplaris embryos. Green color represents gene expression. Nuclei are labeled with DAPI (blue). Images that share the same letter represent data from the same embryo. Anterior is towards the top. a Six3 expression. Dashed line outlines the posterior tip. b Wnt4 expression. c, d Wnt5 expression. e Wnt11 expression. f Wnt16B expression. g, h WntA expression. All panels show lateral views of embryos that are facing right except for d and h which are bilateral views showing internal anatomy. a’, b’, e’, f’ show internal anatomy of laterally viewed embryos. b’ Dashed lines outline developing endomesodermal pouches. d, h Dashed line demarcates the boundary between the head and the trunk

Fig. 8

In situ hybridization results for Wnt genes in stage 13 H. exemplaris embryos. Green color represents gene expression. Nuclei are labeled with DAPI (blue). Images that share the same letter represent data from the same embryo. Anterior is towards the top. a, b Wnt4 expression. c, d Wnt5 expression. e, f Wnt16B expression. g, g’ WntA expression. a, c, e, g Lateral view of embryos that are facing right. d, g’ Bilateral views showing external anatomy. f Bilateral view showing internal anatomy. Dashed line demarcates the boundary between the head and the trunk. a, b, c, e, g Dashed lines demarcate segment boundaries. t1–t4 trunk segment 1–trunk segment 4

Fig. 9

In situ hybridization results for Wnt genes in stage 14 H. exemplaris embryos. Green color represents gene expression. Nuclei are labeled with DAPI (blue). Images that share the same letter represent data from the same embryo. Arrows point to the developing pharynx. Asterisks mark the region between the posteriormost legs. Anterior is towards the top. a, b Wnt5 expression. a Ventral surface of legs is in view. b, b’ Dorsal surface of legs is toward the outside and the ventral surface is toward the inside of the embryo. c, d Wnt11 expression. d Bilateral view. The posterior end of embryo is outlined. e, f Wnt16B expression. g WntA expression. a–b’’, e, f’, f’’ Dashed lines outline legs. a, c, e, g Lateral view of embryos that are facing right. b–b’’, f–f’’, g’ Bilateral view showing internal anatomy. g, g’ Dashed lines demarcate the boundary between the head and the trunk. l1–l4 leg 1–leg 4

Fig. 10

Summary of distribution of Wnt orthologs in metazoan genomes. “X” in white boxes indicates the loss of a Wnt gene. “?” in white boxes indicates that a fully sequenced genome is unavailable for the associated lineage, so it is unclear whether the ortholog has been lost, or is present, but unsequenced. Gray boxes associated with Hofstenia indicate unclear orthology of the four Wnt genes found in the genomes of representatives of this lineage. Dashed lines coming off of Hofstenia represent different hypotheses of the relationship of this lineage with other bilaterians. The interrelationships of panarthropod phyla (Arthropoda, Onychophora, Tardigrada) are depicted as a polytomy because they are not currently resolved [69]. Arthropoda = Drosophila–Ixodes; Onychophora = Euperipatoides; Hypsibius–Ramazzottius = Tardigrada