Ancient origins of axial patterning genes: Hox genes and ParaHox genes in the Cnidaria

Among the bilaterally symmetrical, triploblastic animals (the Bilateria), a conserved set of developmental regulatory genes are known to function in patterning the anterior–posterior (AP) axis. This set includes the well-studied Hox cluster genes, and the recently described genes of the ParaHox cluster, which is believed to be the evolutionary sister of the Hox cluster ( Brooke et al. 1998 ). The conserved role of these axial patterning genes in animals as diverse as frogs and flies is believed to reflect an underlying homology (i.e., all bilaterians derive from a common ancestor which possessed an AP axis and the developmental mechanisms responsible for patterning the axis). However, the origin and early evolution of Hox genes and ParaHox genes remain obscure. Repeated attempts have been made to reconstruct the early evolution of Hox genes by analyzing data from the triphoblastic animals, the Bilateria ( Schubert et al. 1993 ; Zhang and Nei 1996 ). A more precise dating of Hox origins has been elusive due to a lack of sufficient information from outgroup taxa such as the phylum Cnidaria (corals, hydras, jellyfishes, and sea anemones). In combination with outgroup taxa, another potential source of information about Hox origins is outgroup genes (e.g., the genes of the ParaHox cluster). In this article, we present cDNA sequences of two Hox-like genes ( anthox2 and anthox6 ) from the sea anemone, Nematostella vectensis. Phylogenetic analysis indicates that anthox2 (=Cnox2) is homologous to the GSX class of ParaHox genes, and anthox6 is homologous to the anterior class of Hox genes. Therefore, the origin of Hox genes and ParaHox genes occurred prior to the evolutionary split between the Cnidaria and the Bilateria and predated the evolution of the anterior–posterior axis of bilaterian animals. Our analysis also suggests that the central Hox class was invented in the bilaterian lineage, subsequent to their split from the Cnidaria.     

The origins of axial patterning in the metazoa: how old is bilateral symmetry?

Bilateral symmetry is a hallmark of the Bilateria. It is achieved by the intersection of two orthogonal axes of polarity: the anterior-posterior (A-P) axis and the dorsal-ventral (D-V) axis. It is widely thought that bilateral symmetry evolved in the common ancestor of the Bilateria. However, it has long been known that members of the phylum Cnidaria, an outgroup to the Bilateria, also exhibit bilateral symmetry. Recent studies have examined the developmental expression of axial patterning genes in members of the phylum Cnidaria. Hox genes play a conserved role in patterning the A-P axis of bilaterians. Hox genes are expressed in staggered axial domains along the oral-aboral axis of cnidarians, suggesting that Hox patterning of the primary body axis was already present in the cnidarian-bilaterian ancestor. Dpp plays a conserved role patterning the D-V axis of bilaterians. Asymmetric expression of dpp about the directive axis of cnidarians implies that this patterning system is similarly ancient. Taken together, these result imply that bilateral symmetry had already evolved before the Cnidaria diverged from the Bilateria.     

Axial patterning and diversification in the cnidaria predate the Hox system

Across the animal kingdom, Hox genes are organized in clusters whose genomic organization reflects their central roles in patterning along the anterior/posterior (A/P) axis . While a cluster of Hox genes was present in the bilaterian common ancestor, the origins of this system remain unclear (cf. ). With new data for two representatives of the closest extant phylum to the Bilateria, the sea anemone Nematostella and the hydromedusa Eleutheria, we argue here that the Cnidaria predate the evolution of the Hox system. Although Hox-like genes are present in a range of cnidarians, many of these are paralogs and in neither Nematostella nor Eleutheria is an equivalent of the Hox cluster present. With the exception of independently duplicated genes, the cnidarian genes are unlinked and in several cases are flanked by non-Hox genes. Furthermore, the cnidarian genes are expressed in patterns that are inconsistent with the Hox paradigm. We conclude that the Cnidaria/Bilateria split occurred before a definitive Hox system developed. The spectacular variety in morphological and developmental characteristics shown by extant cnidarians demonstrates that there is no obligate link between the Hox system and morphological diversity in the animal kingdom and that a canonical Hox system is not mandatory for axial patterning.     

New ideas on the origin of bilateral animals

Comparative anatomy and embryology provide impressive evidence that the ventral side of all Bilateria (except Chordata) originates from the blastoporal surface, while the mouth and anus develop, respectively, from the anterior and posterior extremities of an elongated blastopore. From the point of view of paleontology, some Vendian multicellular animals represent transitional forms between Radiata and Bilateria. Vendian Bilateria are metameric organisms with a symmetrical or asymmetrical arrangement of segments; they can be considered as bilaterally symmetrical coelenterates crawling on the oral surface. In the recent Cnidaria, homologues of the genes “Brachyury,” “goosecoid” and “fork head” are expressed around the mouth. In the recent Bilateria these genes are expressed along the elongated blastopore and around the mouth and anus. These data corroborate the validity of the idea of amphistomy and the homology between the ventral surface in Bilateria and oral disk in coelenterates. It is supposed that the ancestors of Bilateria were crawling on the oral surface (=ventral side) and gave rise to both Fanerozoic Cnidaria and triploblastic Bilateria. This allows us to suggest the origin of Bilateria from Vendian bilaterally symmetrical coelenterates with numerous metameric pockets of the gastral cavity. Such ancestors gave rise to both Cnidaria and Bilateria. Apparently the primary Bilateria were complicated organisms having a coelom and segmentation, which allows us to explain the great diversity of highly organized organisms (arthropods, mollusks, and others) in the Cambrian era. An idea is proposed that Ctenophora are the only group of recent Eumetazoa that retain primary axial symmetry.     

Asymmetric expression of the BMP antagonists chordin and gremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning

The evolutionary origin of the anterior-posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. Here, we cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral-aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral-aboral axis. We conclude that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral-aboral body axis of Nematostella and one particular body axis of Bilateria.     

Larval development in Cnidaria: A connection to bilateria?

Among the basal animal phyla, the Cnidaria display many characteristics similar to the Bilateria (the higher Metazoa). However, the relation of that outgroup phyla to the Bilateria is still equivocal. Additionally to morphological and genetic data, studies on cnidarian embryogenesis are essential to clarify the Cnidaria-Bilateria relationship. We analyzed cellular differentiation during planula larvae development of the jellyfish Podocoryne carnea. Within 24 to 30 h postfertilization, the diploblastic body structure and all cell types found in polyps have already differentiated in the larva. Whereas the differentiating smooth muscles, RFamide-positive nerve cells, or nematocytes (stinging cells) express no axial polarity, a newly discovered tyrosine-tubulin-positive nervous system develops gradually in repetitive patterns from anterior to posterior. These data demonstrate that part of the cnidarian nervous system develops from anterior to posterior in serially repeated patterns. This developmental mechanism seems to follow the bilaterian pattern and would have antedated the Cambrian explosion.     

WNT unrelated activities in commercially available preparations of recombinant WNT3a

WNT signaling pathways play an important role in both development and disease. By analyzing the signaling capabilities of commercially available WNT3a preparations towards the PI3K/AKT/GSK3 signaling pathway, we discovered unexpected inconsistencies from lot to lot of recombinant WNT3a. We provide evidence that: (1) The ability to trigger AKT/GSK3 signaling varies dramatically between different lots of WNT3a, without any variation in their ability to activate the canonical WNT/β‐catenin signaling. (2) sFRP1, a WNT signaling inhibitor, is unable to interfere with the activation of AKT/GSK3 signaling induced by some of the WNT3a lots. (3) Pharmacological inhibition of AKT/GSK3 phosphorylation by PI3K inhibitors fails to affect the stabilization of β‐catenin, the central effector of the canonical WNT/β‐catenin signaling pathway. In summary, while all tested lots of recombinant WNT3a activated WNT/β‐catenin pathway, our results suggest that individual lots of recombinant WNT3a activate the PI3K/AKT/GSK3 pathway in a WNT‐independent manner, hampering thus the analysis of regulation of PI3K/AKT/GSK3 by WNT ligand. J. Cell. Biochem. 111: 1077–1079, 2010. © 2010 Wiley‐Liss, Inc.     

A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β‐catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here, we report the generation and characterization of a new knock‐in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi‐cistronic targeting cassette at the 3′ end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock‐in allele expresses a bright fluorescent reporter (3xNLS‐SGFP2) and a doxycycline‐inducible driver for lineage tracing (rtTA3). We show that the Axin2^{P2A‐rtTA3‐T2A‐3xNLS‐SGFP2} strain labels WNT/CTNNB1 responsive cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.     

Coordinated regulation of the dorsal‐ventral and anterior‐posterior patterning of Xenopus embryos by the BTB/POZ zinc finger protein Zbtb14

During early vertebrate embryogenesis, bone morphogenetic proteins (BMPs) belonging to the transforming growth factor‐β (TGF‐β) family of growth factors play a central role in dorsal–ventral (DV) patterning of embryos, while other growth factors such as Wnt and fibroblast growth factor (FGF) family members regulate formation of the anterior–posterior (AP) axis. Although the establishment of body plan is thought to require coordinated formation of the DV and AP axes, the mechanistic details underlying this coordination are not well understood. Here, we show that a Xenopus homologue of zbtb14 plays an essential role in the regulation of both DV and AP patterning during early Xenopus development. We show that overexpression of Zbtb14 promotes neural induction and inhibits epidermal differentiation, thereby regulating DV patterning. In addition, Zbtb14 promotes the formation of posterior neural tissue and suppresses anterior neural development. Consistent with this, knock‐down experiments show that Zbtb14 is required for neural development, especially for the formation of posterior neural tissues. Mechanistically, Zbtb14 reduces the levels of phosphorylated Smad1/5/8 to suppress BMP signaling and induces an accumulation of β‐Catenin to promote Wnt signaling. Collectively, these results suggest that Zbtb14 plays a crucial role in the formation of DV and AP axes by regulating both the BMP and Wnt signaling pathways during early Xenopus embryogenesis.     

Resveratrol promotes trophoblast invasion in pre‐eclampsia by inducing epithelial‐mesenchymal transition

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐l ‐arginine methyl ester (L‐NAME). Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.     

MicroRNA‐1275 induces radiosensitization in oesophageal cancer by regulating epithelial‐to‐mesenchymal transition via Wnt/β‐catenin pathway

Acquired radioresistance is one of the main obstacles for the anti‐tumour efficacy of radiotherapy in oesophageal cancer (EC). Recent studies have proposed microRNAs (miRNAs) as important participators in the development of radioresistance in various cancers. Here, we investigated the role of miR‐1275 in acquired radioresistance and epithelial‐mesenchymal transition (EMT) in EC. Firstly, a radioresistant cell line KYSE‐150R was established, with an interesting discovery was observed that miR‐1275 was down‐regulated in KYSE‐150R cells compared to the parental cells. Functionally, miR‐1275 inhibition elevated radioresistance in KYSE‐150 cells via promoting EMT, whereas enforced expression of miR‐1275 increased radiosensitivity in KYSE‐150R cells by inhibiting EMT. Mechanically, we demonstrated that miR‐1275 directly targeted WNT1 and therefore inactivated Wnt/β‐catenin signalling pathway in EC cells. Furthermore, WNT1 depletion countervailed the promoting effect of miR‐1275 suppression on KYSE‐150 cell radioresistance through hampering EMT, whereas WNT1 overexpression rescued miR‐1275 up‐regulation‐impaired EMT to reduce the sensitivity of KYSE‐150R cells to radiation. Collectively, our findings suggested that miR‐1275 suppressed EMT to encourage radiosensitivity in EC cells via targeting WNT1‐activated Wnt/β‐catenin signalling, providing a new therapeutic outlet for overcoming radioresistance of patients with EC.     

Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms

As a sister group to Bilateria, Cnidaria is important for understanding early nervous system evolution. Here we examine neural development in the anthozoan cnidarian Nematostella vectensis in order to better understand whether similar developmental mechanisms are utilized to establish the strikingly different overall organization of bilaterian and cnidarian nervous systems. We generated a neuron-specific transgenic NvElav1 reporter line of N. vectensis and used it in combination with immunohistochemistry against neuropeptides, in situ hybridization and confocal microscopy to analyze nervous system formation in this cnidarian model organism in detail. We show that the development of neurons commences in the ectoderm during gastrulation and involves interkinetic nuclear migration. Transplantation experiments reveal that sensory and ganglion cells are autonomously generated by the ectoderm. In contrast to bilaterians, neurons are also generated throughout the endoderm during planula stages. Morpholino-mediated gene knockdown shows that the development of a subset of ectodermal neurons requires NvElav1, the ortholog to bilaterian neural elav1 genes. The orientation of ectodermal neurites changes during planula development from longitudinal (in early-born neurons) to transverse (in late-born neurons), whereas endodermal neurites can grow in both orientations at any stage. Our findings imply that elav1-dependent ectodermal neurogenesis evolved prior to the divergence of Cnidaria and Bilateria. Moreover, they suggest that, in contrast to bilaterians, almost the entire ectoderm and endoderm of the body column of Nematostella planulae have neurogenic potential and that the establishment of connectivity in its seemingly simple nervous system involves multiple neurite guidance systems.     

A gene trap mutagenesis screen for genes underlying cellular response to the mood stabilizer lithium

Identifying the biological pathways mediating the action of a therapeutic compound may help the development of more specific treatments while also increasing our understanding of the underlying disease pathology. Salts of the metal lithium are commonly used as a front‐line mood stabilizing treatment for bipolar disorder. Lithium's action has been variously linked to inositol phosphate metabolism and the WNT/Glycogen Synthase Kinase 3β (GSK3β)/β‐Catenin signalling cascade, but, to date, little is known about which of these provides the principal therapeutic benefit for patients and, more specifically, which constituent genes, through presumed sequence variation, determine differences in patient response to treatment. Here, we describe a functional screen in which SH‐SY5Y neuroblastoma cells were randomly mutated through genomic integration of the pMS1 poly A ‘gene trap’ plasmid vector. Lithium normally induces differentiation of neuroblastoma cells, but a small proportion of mutated cells continued to proliferate and formed colonies. Rapid amplification of cDNA ends (RACE)‐PCR was used to identify the ‘trapped’ gene in each of these lithium‐resistant colonies. Heterozygous, gene trap integrations were identified within ten genes, eight of which are likely to produce loss‐of‐function mutations including MED10, MSI2 and three long intergenic non‐coding (LINC) RNAs. Both MED10 and MSI2 have been previously linked with WNT/GSK3β/β‐Catenin pathway function suggesting that this is an important mediator of lithium action in this screen. The methodology applied here provides a rapid, objective and economic approach to define the genetic contribution to drug action, but could also be readily adapted to any desired in vitro functional selection/screening paradigm.     

Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling

Primary open‐angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell‐cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out‐flow is largely unknown. The study objective was to investigate the effects of EVs derived from non‐pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome‐mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3β phosphorylation and decreased cytosolic levels of β‐catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKβ regulator‐AKT protein but increased the levels of GSKβ negative regulator‐PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/β‐catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR‐29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR‐29b can be responsible for decreased levels of WNT/β‐catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.