Hox and Wnt pattern the primary body axis of an anthozoan cnidarian before gastrulation

Abstract

Hox gene transcription factors are important regulators of positional identity along the anterior-posterior axis in bilaterian animals. Cnidarians (e.g., sea anemones, corals, and hydroids) are the sister group to the Bilateria and possess genes related to both anterior and central/posterior class Hox genes. Here we report a previously unrecognized domain of Hox expression in the starlet sea anemone, Nematostella vectensis, beginning at early blastula stages. We explore the relationship of two opposing Hox genes (NvAx6/NvAx1) expressed on each side of the blastula during early development. Functional perturbation reveals that NvAx6 and NvAx1 not only regulate their respective expression domains, but also interact with Wnt genes to pattern the entire oral-aboral axis. These findings suggest an ancient link between Hox/Wnt patterning during axis formation and indicate that oral-aboral domains are likely established during blastula formation in anthozoan cnidarians.

Fig. 1

Anterior–posterior patterning and the emergence of a Hox cluster. a Bilaterians are classically defined by an anterior–posterior axis perpendicular to the dorsal ventral axis. Cnidarians are the sister taxa to bilaterians and are the only basal lineage to have a diverse cluster of Hox genes. b The common ancestor of the deuterostome lineage likely had a Hox cluster consisting of 14–15 Hox genes, closely associated with the homeobox gene Eve. c Evidence from the protostome, Tribolium castaneum, suggests that the protostome ancestor also had an intact Hox cluster consisting of at least 10 linked Hox genes^,. d The cnidarian ancestor had both anterior (Hox1 and Hox2) and central/posterior (Hox9–13) class Hox genes. e The Hox complement of the anthozoan cnidarian, Nematostella vectensis, has phylogenetically anterior (NvAx6, NvAx6a, NvAx7, and NvAx8) and central/posterior (NvAx1 and NvAx1a) Hox genes^,. Depiction of Hox expression along the oral–aboral axis of a cnidarian, and the anterior–posterior axis of invertebrates and vertebrates. The anterior (NvAx6) and central/posterior (NvAx1) Hox genes of Nematostella are expressed along the oral–aboral axis during larval development. Regions of anterior, central, and posterior Hox expression are designated with shades of red, green, and blue, respectively. Asterisk indicates site of mouth formation

Fig. 2

Hox genes of Nematostella exhibit hallmarks of bilaterian Hox genes during early development. a Time series of the embryonic, larval, and adult development of the cnidarian, Nematostella vectensis (hours post fertilization = hpf). b Clustered Hox genes display temporal colinearity during early embryonic development. Expression begins with the 3′ located neighboring non-Hox homeobox gene NvHlxB9 (data not shown) and the anterior gene NvAx6 at 12hpf. Subsequent activation of the other genes in the cluster maintains colinear expression relative to the ancestral cnidarian cluster. Two of the unlinked Hox genes NvAx1 (central/posterior) and NvAx6a (anterior) appear maternally expressed during early development. c In situ hybridization confirms the temporal activation of the Hox cluster. Prior to gastrulation, mRNA expression of the central/posterior Hox gene (NvAx1) localizes to the aboral pole, while an anterior Hox gene (NvAx6) becomes transcriptionally activated along the oral pole. Remaining Hox and homeobox genes occupy oral (NvEve, NvAv7, NvAx6a) or aboral (NvAx8) domains during early development. Images were compiled from at least three separate experiments. Error bars are based on S.E.M. Scale bars are 50 µm

Fig. 3

Perturbation of Hox expression disrupts oral patterning during gastrulation. Developmental time series of anterior, NvAx6 (a–e), and central/posterior, NvAx1 (f–j), Hox mRNA expression at blastula (a, f), early gastrula (b, g), mid gastrula (c, h), early planula (d, i), and late planula stages (e, j; images reproduced from ref.). k–o Gastrulation defects due to disruption of anterior (NvAx6) and central/posterior (NvAx1) Hox expression through microinjection of antisense translation blocking morpholinos (knockdown) or in vitro transcribed mRNA (overexpression). k Fluorescent phalloidin-labeled embryo during final stages of gastrulation (48hpf) with distinct ectodermal(Ec) and endo-mesodermal(En) tissue layers delineated by the early pharynx (P) (white-dashed line delineates the pharynx from endomesoderm). l Knockdown of anterior Hox (NvAx6) blocks invagination of the presumptive endomesoderm. m Overexpression of central/posterior Hox (NvAx1) mRNA also blocks gastrulation and produces gastrula stage embryos with reduced axial morphology. n Overexpression of anterior Hox (NvAx6) mRNA and o knockdown of the central/posterior Hox gene (NvAx1) disrupts pharynx development and produces ectopic oral tissue at the blastopore (white arrowhead). p–r In situ hybridization of anterior Hox (NvAx6) at 24hpf in control (p), central/posterior Hox knockdown (q), and central/posterior Hox overexpression treatments (r). s–u In situ hybridization of central/posterior Hox (NvAx1) at 24hpf in control (s), anterior Hox knockdown (t), and anterior Hox overexpression treatments (u). Images were compiled from at least three separate experiments and the number of similar phenotypes is noted as a fraction in the lower left-hand corner (e.g., k—77 out of 80 total animals exhibited the phenotype). Scale bars are 50 µm

Fig. 4

Effects of perturbing anterior and central/posterior Hox genes on oral–aboral identity extends to late development. a–c Phalloidin stained embryos at planula larvae (144hpf) stages taken from: a dextran-injected controls, b anterior Hox (NvAx6) overexpression, and c central/posterior Hox (NvAx1) overexpression treatments. d–h Apical tuft cilia labeled with and acetylated tubulin antibody (red) with the nuclei counter stained with DAPI (blue) in: d planula stage embryos treated with dextran, e anterior NvAx6 morpholino, f central/posterior NvAx1 Hox mRNA, g anterior NvAx6 mRNA, or h central/posterior NvAx1 Hox morpholino. i–l Changes in polyp morphology resulting from disruption of anterior Hox (NvAx6) and central/posterior Hox (NvAx1) expression during early development. i Wild-type polyp after metamorphosis. j Overexpression of anterior Hox (NvAx6) results in ectopic tissue formation at the oral side of the animal. k Overexpression of central/posterior Hox (NvAx1) results in bilayered animals (maintained for 21 days after fertilization) without clear axial morphology but have cnidocytes (stinging cells) along the outer ectoderm. l Animals treated with central/posterior Hox (NvAx1) morpholino eventually metamorphose (2 weeks after fertilization) to produce animals with small heads and extremely long body columns. Images were compiled from at least three separate experiments and the number of similar phenotypes is noted as a fraction in the lower left-hand corner. Scale bars are 50 µm

Fig. 5

Anterior and central/posterior Hox genes have reciprocal phenotypes during oral but not aboral development. a Schematic representation of perturbation of either anterior Hox (NvAx6) or central/posterior Hox (Ax1) during early development. a, b Late gastrula (48hpf) stage expression of molecular markers for oral (a) or aboral (b) territories. White-dashed lines in NvFoxA column serve to highlight the larval pharynx and distinguish between the ectoderm and endoderm. White-dashed lines in the NvWnt2 column outline the belt of NvWnt2 expression. c–g Illustration summarizing territorial changes due to manipulation of anterior (NvAx6) and central/posterior (NvAx1) Hox genes. Images were compiled from at least three separate experiments and the number of similar phenotypes is noted as a fraction in the lower left-hand corner. Scale bars are 50 µm

Fig. 6

Loss of anterior and central/posterior Hox results in loss of oral–aboral patterning, but not endomesoderm formation. Gastrulation defects resulting from co-injection experiments as assessed by fluorescent phalloidin labeling (far left column) and in situ hybridization for oral (NvSnailA, NvBrachyury, and NvFoxA) and aboral (NvFgf2a, NvSfrp1/5, and NvSix3/6) markers. Ec ectoderm; En endo-mesodermal; and P pharynx. White-dashed line serves to distinguish the point of transition between the ectoderm and the endoderm. Images were compiled from at least three separate experiments and the number of similar phenotypes is noted as a fraction in the lower left-hand corner

Fig. 7

Model of Hox gene function in early development of an anthozoan. a Illustration depicting anterior Hox (NvAx6) (red) and central/posterior Hox (NvAx1) (blue/green) expression throughout early development of N. vectensis. b Working model of how anterior (NvAx6) and central/posterior (NvAx1) Hox genes function to specify the oral–aboral axis before and during gastrulation in an anthozoan cnidarian. c Illustration of known regulatory relationships between Hox genes and the oral (NvWntA, NvWnt1, NvWnt2, NvWnt3, NvWnt4, NvFoxA, NvBrachyury, and NvSnailA) and aboral (NvSix3/6, NvFgf2A, NvSfrp1/5, and NvDkk1/2/4) markers assessed in this study. Important components of this regulatory paradigm needing description are marked with roman numerals (I–IX) with corresponding text at the periphery of the illustration. Regulatory relationships derived from previous studies are cited in the periphery text (I, II, VI, and VIII)