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HOX gene complement and expression in the planarian Schmidtea mediterranea - PubMed

  • ️Fri Jan 01 2016

HOX gene complement and expression in the planarian Schmidtea mediterranea

Ko W Currie et al. Evodevo. 2016.

Abstract

Background: Freshwater planarians are well known for their regenerative abilities. Less well known is how planarians maintain spatial patterning in long-lived adult animals or how they re-pattern tissues during regeneration. HOX genes are good candidates to regulate planarian spatial patterning, yet the full complement or genomic clustering of planarian HOX genes has not yet been described, primarily because only a few have been detectable by in situ hybridization, and none have given morphological phenotypes when knocked down by RNAi.

Results: Because the planarian Schmidtea mediterranea (S. mediterranea) is unsegmented, appendage less, and morphologically simple, it has been proposed that it may have a simplified HOX gene complement. Here, we argue against this hypothesis and show that S. mediterranea has a total of 13 HOX genes, which represent homologs to all major axial categories, and can be detected by whole-mount in situ hybridization using a highly sensitive method. In addition, we show that planarian HOX genes do not cluster in the genome, yet 5/13 have retained aspects of axially restricted expression. Finally, we confirm HOX gene axial expression by RNA deep-sequencing 6 anterior-posterior "zones" of the animal, which we provide as a dataset to the community to discover other axially restricted transcripts.

Conclusions: Freshwater planarians have an unappreciated HOX gene complexity, with all major axial categories represented. However, we conclude based on adult expression patterns that planarians have a derived body plan and their asexual lifestyle may have allowed for large changes in HOX expression from the last common ancestor between arthropods, flatworms, and vertebrates. Using our in situ method and axial zone RNAseq data, it should be possible to further understand the pathways that pattern the anterior-posterior axis of adult planarians.

Keywords: Body plan; Flatworm; HOX; Homeotic; In situ hybridization; Lophotrochozoan; Planarians; Schmidtea mediterranea.

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Figures

Fig. 1
Fig. 1

HOX complement and phylogenetic assignments in S. mediterranea. A Bayesian phylogeny of select animal HOX genes as well as caudal (Cdx), even-skipped (Evx), engrailed (En), Gsx/ind, and Xlox is shown. Genes from S. mediterranea are boxed in red. Two genes from S. mediterranea could not be placed with confidence: HOX3b and HOX4b, which are marked with red asterisks. Of note, S. mediterranea has 4 Post-2 paralogs and no Cdx, Gsx, or Xlox genes. The tree used the engrailed sub-tree as an outgroup, and only the posterior probabilities are shown for important nodes (maximum likelihood tree shown in Additional file 2: Fig. S3). Species and sequences used in the phylogeny are listed in Additional file 1: Table S1. Briefly: Smed = Schmidtea mediterranea; Ctel = Capitella teleta (polychaete annelid); Amph = Amphioxus (cephalocordate; mix of Branchiostoma species floridae and lanceolatum); Tcas = Tribolium castaneum (flour beetle); Latk = Lithobius atkinsoni (centipede); Mlig = Macrostomum lignano (basal flatworm); Smar = Strigamia maritima (centipede); Pdum = Platynereis dumerilii (polychaete annelid); Erub = Ethmostigmus rubripes (giant centipede); Hrob = Helobdella robusta (leech, annelid); Lgig = Lottia gigantea (owl limpet mollusk); Amel = Apis mellifera (honeybee); Xtro = Xenopus tropicalis (frog); Atyp = Archaster typicus (starfish, hemichordate); Esco = Euprymna scolopes (bobtail squid mollusk)

Fig. 2
Fig. 2

RNAseq and WISH reveal axially restricted HOX genes. a A cartoon schematic of 6 axial zones of tissue subjected to RNAseq is shown at the left. b Following transcript normalization across all zones, transcripts were binned according to having specific expression in particular zones. Twenty-two binning categories were distinguished, of which 16 contain specificity to more than one zone. For example, a specificity of 1.2 would signify that there was specificity to zones 1 and 2, with higher expression in zone 1 (whereas 2.1 has the same specificity but higher expression in zone 2). All transcripts and categories are listed in Additional file 3: Table S2. c–p FISH stains for individual HOX genes are shown in magenta and counterstained with DAPI in gray. Arrows denote where the strongest area of expression was seen. For each gene, the raw CPM values per zone are given in the histogram to the right of each stain. Two main categories were observed: axial restriction perpendicular to the A–P axis (c–l) and radial expression inside of the body edge at the D-V boundary (m–p)

Fig. 3
Fig. 3

Tissue-specific expression of the HOX genes. For HOX genes that did not show axial restriction by WISH or RNAseq in uninjured animals, all others were detected in specific tissues. Most often, brain and nerve cord expression was detected and confirmed by dFISH with the marker chat in green to mark cholinergic neurons (a–l). Post-2b showed detectable expression in stem cells using the marker piwi-1 (m, n), and Post-1 was found to be expressed in a small subset of piwi-1 + cells that are in the anatomical location of primordial germ cells in asexual planarians (o, p) [71, 72]. The remaining genes were found to be expressed in the epithelium (q, r), the body opening (s, t), the pharynx (u), as well as unknown mesenchymal cells with broad distribution (v, w). Scale bars = 50 μm

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