Wnt signaling is required for antero-posterior patterning of the planarian brain

Wnt signaling functions in axis formation and morphogenesis in various animals and organs. Here we report that Wnt signaling is required for proper brain patterning during planarian brain regeneration. We showed here that one of the Wnt homologues in the planarian Dugesia japonica, DjwntA, was expressed in the posterior region of the brain. When DjwntA-knockdown planarians were produced by RNAi, they could regenerate their heads at the anterior ends of the fragments, but formed ectopic eyes with irregular posterior lateral branches and brain expansion. This suggests that the Wnt signal may be involved in antero-posterior (A-P) patterning of the planarian brain, as in vertebrates. We also investigated the relationship between the DjwntA and nou-darake/FGFR signal systems, as knockdown planarians of these genes showed similar phenotypes. Double-knockdown planarians of these genes did not show any synergistic effects, suggesting that the two signal systems function independently in the process of brain regeneration, which accords with the fact that nou-darake was expressed earlier than DjwntA during brain regeneration. These observations suggest that the nou-darake/FGFR signal may be involved in brain rudiment formation during the early stage of head regeneration, and subsequently the DjwntA signal may function in A-P patterning of the brain rudiment.     

Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo

In Xenopus, the Mix/Bix family of homeobox genes has been implicated in mesendoderm development. Mixl1 is the only known murine member of this family. To examine the role of Mixl1 in murine embryogenesis, we used gene targeting to create mice bearing a null mutation of Mixl1. Homozygous Mixl1 mutant embryos can be distinguished from their littermates by a marked thickening of the primitive streak. By the early somite stage, embryonic development is arrested, with the formation of abnormal head folds, foreshortened body axis, absence of heart tube and gut, deficient paraxial mesoderm, and an enlarged midline tissue mass that replaces the notochord. Development of extra-embryonic structures is generally normal except that the allantois is often disproportionately large for the size of the mutant embryo. In chimeras, Mixl1(-/-) mutant cells can contribute to all embryonic structures, with the exception of the hindgut, suggesting that Mixl1 activity is most crucial for endodermal differentiation. Mixl1 is therefore required for the morphogenesis of axial mesoderm, the heart and the gut during embryogenesis.     

A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.     

A heat shock protein and Wnt signaling crosstalk during axial patterning and stem cell proliferation

Both Wnt signaling and heat shock proteins play important roles in development and disease. As such, they have been widely, though separately, studied. Here we show a link between a heat shock protein and Wnt signaling in a member of the basal phylum, Cnidaria. A heat shock at late gastrulation in the clonal marine hydrozoan, Hydractinia, interferes with axis development, specifically inhibiting head development, while aboral structures remain unaffected. The heat treatment upregulated Hsc71, a constitutive Hsp70 related gene, followed by a transient upregulation, and long-term downregulation, of Wnt signaling components. Downregulating Hsc71 by RNAi in heat-shocked animals rescued these defects, resulting in normal head development. Transgenic animals, ectopically expressing Hsc71, had similar developmental abnormalities as heat-shocked animals in terms of both morphology and Wnt3 expression. We also found that Hsc71 is upregulated in response to ectopic Wnt activation, but only in the context of stem cell proliferation and not in head development. Hsc71's normal expression is consistent with a conserved role in mitosis and apoptosis inhibition. Our results demonstrate a hitherto unknown crosstalk between heat shock proteins and Wnt/β-catenin signaling. This link likely has important implications in understanding normal development, congenital defects and cancer biology.     

Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.     

Daam2 is required for dorsal patterning via modulation of canonical Wnt signaling in the developing spinal cord

The Daam family of proteins consists of Daam1 and Daam2. Although Daam1 participates in noncanonical Wnt signaling during gastrulation, Daam2 function remains completely uncharacterized. Here we describe the role of Daam2 in canonical Wnt signal transduction during spinal cord development. Loss-of-function studies revealed that Daam2 is required for dorsal progenitor identities and canonical Wnt signaling. These phenotypes are rescued by β-catenin, demonstrating that Daam2 functions in dorsal patterning through the canonical Wnt pathway. Complementary gain-of-function studies demonstrate that Daam2 amplifies Wnt signaling by potentiating ligand activation. Biochemical examination found that Daam2 association with Dvl3 is required for Wnt activity and dorsal patterning. Moreover, Daam2 stabilizes Dvl3/Axin2 binding, resulting in enhanced intracellular assembly of Dvl3/Axin2 complexes. These studies demonstrate that Daam2 modulates the formation of Wnt receptor complexes, revealing new insight into the functional diversity of Daam proteins and how canonical Wnt signaling contributes to pattern formation in the developing spinal cord.     

A role for MKP3 in axial patterning of the zebrafish embryo

Fibroblast growth factors (FGFs) are secreted molecules that can activate the RAS/mitogen-activated protein kinase (MAPK) pathway to serve crucial functions during embryogenesis. Through an in situ hybridization screen for genes with restricted expression patterns during early zebrafish development, we identified a group of genes that exhibit similar expression patterns to FGF genes. We report the characterization of zebrafish MAP kinase phosphatase 3 (MKP3; DUSP6 - Zebrafish Information Network), a member of the FGF synexpression group, showing that it has a crucial role in the specification of axial polarity in the early zebrafish embryo. MKP3 dephosphorylates the activated form of MAPK, inhibiting the RAS/MAPK arm of the FGF signaling pathway. Gain- and loss-of-function studies reveal that MKP3 is required to limit the extent of FGF/RAS/MAPK signaling in the early embryo, and that disturbing this inhibitory pathway disrupts dorsoventral patterning at the onset of gastrulation. The earliest mkp3 expression is restricted to the future dorsal region of the embryo where it is initiated by a maternal beta-catenin signal, but soon after its initiation, mkp3 expression comes under the control of FGF signaling. Thus, mkp3 encodes a feedback attenuator of the FGF pathway, the expression of which is initiated at an early stage so as to ensure correct FGF signaling levels at the time of axial patterning.     

Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)

Nematogenesis, the production of stinging cells (nematocytes) in Cnidaria, can be considered as a model neurogenic process. Most molecular data concern the freshwater polyp Hydra, in which nematocyte production is scattered throughout the body column ectoderm, the mature cells then migrating to the tentacles. We have characterized tentacular nematogenesis in the Clytia hemisphaerica hydromedusa and found it to be confined to the ectoderm of the tentacle bulb, a specialized swelling at the tentacle base. Analysis by a variety of light and electron microscope techniques revealed that while cellular aspects of nematogenesis are similar to Hydra, the spatio-temporal characteristics are markedly more ordered. The tentacle bulb nematogenic ectoderm (TBE) was found to be polarized, with a clear progression of successive nematoblast stages from a proximal zone (comprising a majority of undifferentiated cells) to the distal end where the tentacle starts. Pulse-chase labelling experiments demonstrated a continuous displacement of differentiating nematoblasts towards the tentacle tip, and that nematogenesis proceeds more rapidly in Clytia than in Hydra. Compact expression domains of orthologues of known nematogenesis-associated genes (Piwi, dickkopf-3, minicollagens and NOWA) were correspondingly staggered along the TBE. These distinct characteristics make the Clytia TBE a promising experimental system for understanding the mechanisms regulating nematogenesis.     

Dazap2 is required for FGF-mediated posterior neural patterning, independent of Wnt and Cdx function

The organization of the embryonic neural plate requires coordination of multiple signal transduction pathways, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and WNTs. Many studies have suggested that a critical component of this process is the patterning of posterior neural tissues by an FGF-caudal signaling cascade. Here, we have identified a novel player, Dazap2, and show that it is required in vivo for posterior neural fate. Loss of Dazap2 in embryos resulted in diminished expression of hoxb9 with a concurrent increase in the anterior marker otx2. Furthermore, we found that Dazap2 is required for FGF dependent posterior patterning; surprisingly, this is independent of Cdx activity. Furthermore, in contrast to FGF activity, Dazap2 induction of hoxb9 is not blocked by loss of canonical Wnt signaling. Functionally, we found that increasing Dazap2 levels alters neural patterning and induces posterior neural markers. This activity overcomes the anteriorizing effects of noggin, and is downstream of FGF receptor activation. Our results strongly suggest that Dazap2 is a novel and essential branch of FGF-induced neural patterning.     

A revised fate map for amphioxus and the evolution of axial patterning in chordates

The chordates include vertebrates plus two groups of invertebrates(the cephalochordates and tunicates). Previous embryonic fatemaps of the cephalochordate amphioxus (Branchiostoma) were influencedby preconceptions that early development in amphioxus and ascidiantunicates should be fundamentally the same and that the earlyamphioxus embryo, like that of amphibians, should have ventralmesoderm. Although detailed cell lineage tracing in amphioxushas not been done because of limited availability of the embryosand because cleavage is radial and holoblastic with the blastomeresnearly equal in size and not tightly adherent until the mid-blastulastage, a compilation of data from gene expression and function,blastomere isolation and dye labeling allows a more realisticfate map to be drawn. The revised fate map is substantiallydifferent from that of ascidians. It shows (1) that the anteriorpole of the amphioxus embryo is offset dorsally from the animalpole only by about 20°, (2) that the ectoderm/mesendodermboundary (the future rim of the blastopore) is at the equatorof the blastula, which approximately coincides with the 3rdcleavage plane, and (3) that there is no ventral mesoderm duringthe gastrula stage. Involution or ingression of cells over theblastopore lip is negligible, and the blastopore, which is posterior,closes centripetally as if by a purse string. During the gastrulastage, the animal pole shifts ventrally, coming to lie about20° ventral to the anterior tip of the late gastrula/earlyneurula. Comparisons of the embryos of amphioxus and vertebratesindicate that in spite of large differences in the mechanicsof cleavage and gastrulation, anterior/posterior and dorsal/ventralpatterning occur by homologous genetic mechanisms. Therefore,the small, nonyolky embryo of amphioxus is probably a reasonableapproximation of the basal chordate embryo before the evolutionof determinate cleavage in the tunicates and the evolution largeamounts of yolk in basal vertebrates.     

The establishment of axial patterning in the maize leaf

The maize leaf consists of four distinct tissues along its proximodistal axis: sheath, ligule, auricle and blade. liguleless1 (lg1) functions cell autonomously to specify ligule and auricle, and may propagate a signal that correctly positions the blade-sheath boundary. The dominant Wavy auricle in blade (Wab1) mutation disrupts both the mediolateral and proximodistal axes of the maize leaf. Wab1 leaf blades are narrow and ectopic auricle and sheath extend into the blade. The recessive lg1-R mutation exacerbates the Wab1 phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. We show that lg1 is misexpressed in Wab1 leaves. Our results suggest that the Wab1 defect is partially compensated for by lg1 expression. A mosaic analysis of Wab1 was conducted in Lg1+ and lg1-R backgrounds to determine if Wab1 affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1+/- sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1+ background. These results suggest that lg1 can influence the autonomy of Wab1. In both genotypes, leaf-halves with wab1+/- sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1 acts cell-autonomously to affect lateral growth. The mosaic analysis, lg1 expression data and comparison of mutant leaf shapes reveal previously unreported functions of lg1 in both normal leaf development and in the dominant Wab1 mutant.     

Components of both major axial patterning systems of the Bilateria are differentially expressed along the primary axis of a 'radiate' animal, the anthozoan cnidarian Acropora millepora

Cnidarians are animals with a single (oral/aboral) overt body axis and with origins that nominally predate bilaterality. To better understand the evolution of axial patterning mechanisms, we characterized genes from the coral, Acropora millepora (Class Anthozoa) that are considered to be unambiguous markers of the bilaterian anterior/posterior and dorsal/ventral axes. Homologs of Otx/otd and Emx/ems, definitive anterior markers across the Bilateria, are expressed at opposite ends of the Acropora larva; otxA-Am initially around the blastopore and later preferentially toward the oral end in the ectoderm, and emx-Am predominantly in putative neurons in the aboral half of the planula larva, in a domain overlapping that of cnox-2Am, a Gsh/ind gene. The Acropora homologs of Pax-3/7, NKX2.1/vnd and Msx/msh are expressed in axially restricted and largely non-overlapping patterns in larval ectoderm. In Acropora, components of both the D/V and A/P patterning systems of bilateral animals are therefore expressed in regionally restricted patterns along the single overt body axis of the planula larva, and two 'anterior' markers are expressed at opposite ends of the axis. Thus, although some specific gene functions appear to be conserved between cnidarians and higher animals, no simple relationship exists between axial patterning systems in the two groups.     

Genetic evidence for involvement of maternally derived Wnt canonical signaling in dorsal determination in zebrafish

In zebrafish, the program for dorsal specification begins soon after fertilization. Dorsal determinants are localized initially to the vegetal pole, then transported to the blastoderm, where they are thought to activate the canonical Wnt pathway, which induces the expression of dorsal-specific genes. We identified a novel maternal-effect recessive mutation, tokkaebi (tkk), that affects formation of the dorsal axis. Severely ventralized phenotypes, including a lack of dorso-anterior structures, were seen in 5-100% of the embryos obtained from tkk homozygous transmitting females. tkk embryos displayed defects in the nuclear accumulation of beta-catenin on the dorsal side, and reduced or absent expression of dorsal-specific genes. Mesoderm and endoderm formation outside the dorsal axis was not significantly affected. Injection of RNAs for activated beta-catenin, dominant-negative forms of Axin1 and GSK3beta, and wild-type Dvl3, into the tkk embryos suppressed the ventralized phenotypes and/or dorsalized the embryos, and restored or induced an ectopic and expanded expression of bozozok/dharma and goosecoid. However, dorsalization by wnt RNAs was affected in the tkk embryos. Inhibition of cytoplasmic calcium release elicited an ectopic and expanded expression of chordin in the wild-type, but did not restore chordin expression efficiently in the tkk embryos. These data indicate that the tkk gene product functions upstream of or parallel to the beta-catenin-degradation machinery to control the stability of beta-catenin. The tkk locus was mapped to chromosome 16. These data provide genetic evidence that the maternally derived canonical Wnt pathway upstream of beta-catenin is involved in dorsal axis formation in zebrafish.     

A new L-type calcium channel isoform required for normal patterning of the developing neuromuscular junction

One of the earliest steps in the development of the neuromuscular junction (NMJ) is the pre-patterning of acetylcholine receptors (AChR) in the center of muscle fibers. This process has recently been proposed to depend on L-type calcium currents. But its feeble current properties make the skeletal muscle calcium channel (Ca(v)1.1) a poor candidate for this function. Independently a new Ca(v)1.1e splice variant with greatly distinct current properties has been described. But so far this channel lacked a function. Could this orphan channel be responsible for regulating AChR pre-patterning? Here we compare the properties of the two splice variants and argue that the newly discovered variant Ca(v)1.1e, is dominantly expressed at the proper time in development and has the essential current properties to accomplish the proposed role in the formation of the NMJ.     

A new L-type calcium channel isoform required for normal patterning of the developing neuromuscular junction

One of the earliest steps in the development of the neuromuscular junction (NMJ) is the pre-patterning of acetylcholine receptors (AChR) in the center of muscle fibers. This process has recently been proposed to depend on L-type calcium currents. But its feeble current properties make the skeletal muscle calcium channel (Ca_v1.1) a poor candidate for this function. Independently a new Ca_v1.1e splice variant with greatly distinct current properties has been described. But so far this channel lacked a function. Could this orphan channel be responsible for regulating AChR pre-patterning? Here we compare the properties of the two splice variants and argue that the newly discovered variant Ca_v1.1e, is dominantly expressed at the proper time in development and has the essential current properties to accomplish the proposed role in the formation of the NMJ.