RESUMO
Formation of a constriction and tissue separation between parent and young polyp is a hallmark of the Hydra budding process and controlled by fibroblast growth factor receptor (FGFR) signaling. Appearance of a cluster of cells positive for double phosphorylated ERK (dpERK) at the late separation site indicated that the RAS/MEK/ERK pathway might be a downstream target of the Hydra Kringelchen FGFR. In fact, inhibition of ERK phosphorylation by the MEK inhibitor U0126 reversibly delayed bud detachment and prevented formation of the dpERK-positive cell cluster indicating de novo-phosphorylation of ERK at the late bud base. In functional studies, a dominant-negative Kringelchen FGFR prevented bud detachment as well as appearance of the dpERK-positive cell cluster. Ectopic expression of full length Kringelchen, on the other hand, induced a localized rearrangement of the actin cytoskeleton at sites of constriction, localized ERK-phosphorylation and autotomy of the body column. Our data suggest a model in which (i) the Hydra FGFR targets, via an unknown pathway, the actin cytoskeleton to induce a constriction and (ii) FGFR activates MEK/ERK signaling at the late separation site to allow tissue separation.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hydra/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Animais Geneticamente Modificados , Western Blotting , Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hydra/embriologia , Hydra/crescimento & desenvolvimento , Hibridização In Situ , Microscopia Confocal , Microscopia de Fluorescência , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Pirróis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de TempoRESUMO
Guidance of cells and tissue sheets is an essential function in developing and differentiating animal tissues. In Hydra, where cells and tissue move dynamically due to constant cell proliferation towards the termini or into lateral, vegetative buds, factors essential for guidance are still unknown. Good candidates to take over this function are fibroblast growth factors (FGFs). We present the phylogeny of several Hydra FGFs and analysis of their expression patterns. One of the FGFs is expressed in all terminal regions targeted by tissue movement and at boundaries crossed by moving tissue and cells with an expression pattern slightly differing in two Hydra strains. A model addressing an involvement of this FGF in cell movement and morphogenesis is proposed: Hydra FGFf-expressing cells might serve as sources to attract tissue and cells towards the termini of the body column and across morphological boundaries. Moreover, a function in morphogenesis and/or differentiation of cells and tissue is suggested.
Assuntos
Evolução Molecular , Fatores de Crescimento de Fibroblastos/genética , Hydra/genética , Hydra/metabolismo , Animais , Etiquetas de Sequências Expressas , Fatores de Crescimento de Fibroblastos/metabolismo , Hydra/crescimento & desenvolvimento , Morfogênese , Filogenia , Regulação para CimaRESUMO
In the polychaete Platynereis dumerilii exactly four primordial germ cells (PGCs) arise in early development and are subject to a transient mitotic arrest until the animals enter gametogenesis. In order to unravel the mechanisms controlling the number of PGCs in Platynereis, we tested whether the steroid 17ß-estradiol (E2) is able to induce PGC proliferation, as it had been described in other species. Our data provide strong support for such a mechanism, showing that E2 significantly increases the occurrence of larvae with supernumerary PGCs in Platynereis in a dose dependent manner. E2 responsiveness is restricted to early developmental stages, when the PGCs are specified. During these stages, embryos exhibit high expression levels of the estradiol receptor (ER). The ER transcript localizes to the yolk-free cytoplasm of unfertilized eggs and segregates into the micromeres during cleavage stages. Nuclear ER protein is found asymmetrically distributed between daughter cells. Neither transcript nor protein is detectable in PGCs at larval stages. Addition of the specific estradiol receptor inhibitor ICI-182,780 (ICI) abolishes the proliferative effect of E2, suggesting that it is mediated by ER signaling. Our study reports for the first time an ER mediated proliferative effect of E2 on PGCs in an invertebrate organism.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Células Germinativas/efeitos dos fármacos , Receptores de Estradiol/metabolismo , Animais , Núcleo Celular/metabolismo , Proliferação de Células , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Fulvestranto , Células Germinativas/citologia , Células Germinativas/metabolismo , Técnicas Imunoenzimáticas , Hibridização In Situ , Poliquetos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estradiol/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
Fibroblast growth factor receptors (FGFR) are highly conserved receptor tyrosine kinases, and evolved early in metazoan evolution. In order to investigate their functional conservation, we asked whether the Kringelchen FGFR in the freshwater polyp Hydra vulgaris, is able to functionally replace FGFR in fly embryos. In Drosophila, two endogenous FGFR, Breathless (Btl) and Heartless (Htl), ensure formation of the tracheal system and mesodermal cell migration as well as formation of the heart. Using UAS-kringelchen-5xmyc transgenic flies and targeted expression, we show that Kringelchen is integrated correctly into the cell membrane of mesodermal and tracheal cells in Drosophila. Nevertheless, Kringelchen expression driven in tracheal cells failed to rescue the btl (LG19) mutant. The Hydra FGFR was able to substitute for Heartless in the htl (AB42) null mutant; however, this occurred only during early mesodermal cell migration. Our data provide evidence for functional conservation of this early-diverged FGFR across these distantly related phyla, but also selectivity for the Htl FGFR in the Drosophila system.
Assuntos
Drosophila/genética , Hydra/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Evolução Molecular , Dados de Sequência Molecular , Mutação , Filogenia , Receptores de Fatores de Crescimento de Fibroblastos/química , Homologia de Sequência de AminoácidosRESUMO
Molecular data for nephridial development in polychaetes are not available yet. The scope of our work was to establish a reference system for future investigations using two markers for nephridial development: beta-tubulin as marker for cilia and alkaline phosphatase (AP) activity for secretory epithelia. The markers identified, unexpectedly, three consecutively forming generations of nephridia: (1) a transitory unciliated, but AP-positive head kidney, (2) a transitory larval nephridium, which undergoes a morphological transition from a protonephridium to a funnelled nephridium concomitant with the development of the coelomic cavity and finally, (3) the serially arranged metanephridia. The spatial arrangement of larval and definitive nephridia, revealed an up to now unknown developmental boundary between the synchronously forming larval and the serially proliferating definitive segments. Development of three consecutive sets of nephridia with different morphology and biochemical properties was unexpected and reveals an interesting multistep process in the development of excretory structures in Platynereis.
Assuntos
Larva/crescimento & desenvolvimento , Poliquetos/embriologia , Fosfatase Alcalina/metabolismo , Animais , Imuno-Histoquímica , Larva/metabolismo , Larva/ultraestrutura , Microscopia Eletrônica de Transmissão , Poliquetos/metabolismo , Poliquetos/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa.
Assuntos
Evolução Molecular , Hydra/genética , Placozoa/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Animais , Cnidários/genética , Estrutura Terciária de Proteína , Receptores de Fatores de Crescimento de Fibroblastos/químicaRESUMO
Elucidating the origin of germ cells in embryos and larvae is often obscured by the fact that the typical germ cell markers vasa, nanos and piwi are not exclusively expressed in primordial germ cells (PGCs), but are also commonly found in undifferentiated somatic tissues and stem cells as part of an evolutionary conserved 'germline multipotency program' (Juliano et al., 2010). Hidden in the crowd of undifferentiated cells, the PGCs have occasionally been overlooked and their formation during early embryogenesis was only revealed recently by new methodological approaches (e.g. Wu et al., 2011). Spiralians are excellent model organisms to deepen our understanding of PGC formation, given the highly stereotypical cleavage that occurs during embryogenesis. In these species, detailed cell lineage studies enable the tracing of single cells up to gastrulation stages. Here, I review our knowledge of the origin of PGCs in these invertebrates. Similarities in PGC formation among spiralian phyla as well as peculiarities of the highly derived clitellates are discussed with respect to developmental mode and evolution. Furthermore, the issue of gonad regeneration in platyhelminths and the asexually reproducing oligochaete Enchytraeus japonensis is addressed. An alternative strategy of compensating for caudal regeneration is presented for the polychaete Platynereis dumerilli. Finally, the molecular bases of PGC specification and the question of germplasm are discussed.
Assuntos
Embrião não Mamífero/citologia , Desenvolvimento Embrionário/fisiologia , Larva/crescimento & desenvolvimento , Oligoquetos/embriologia , Poliquetos/embriologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Germinativas/citologia , Oligoquetos/crescimento & desenvolvimento , Poliquetos/crescimento & desenvolvimentoRESUMO
BACKGROUND: In the polychaete Platynereis, the primordial germ cells (PGCs) emerge from the vasa, piwi, and PL10 expressing mesodermal posterior growth zone (MPGZ) at the end of larval development, suggesting a post-embryonic formation from stem cells. METHODS: In order to verify this hypothesis, embryos and larvae were pulse labeled with the proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) at different stages of development. Subsequently, the PGCs were visualized in 7-day-old young worms using antibodies against the Vasa protein. RESULTS: Surprisingly, the primordial germ cells of Platynereis incorporate EdU only shortly before gastrulation (6-8 hours post fertilization (hpf)), which coincides with the emergence of four small blastomeres from the mesoblast lineage. We conclude that these so-called 'secondary mesoblast cells' constitute the definitive PGCs in Platynereis. In contrast, the cells of the MPGZ incorporate EdU only from the pre-trochophore stage onward (14 hpf). CONCLUSION: While PGCs and the cells of the MPGZ in Platynereis are indistinguishable in morphology and both express the germline markers vasa, nanos, and piwi, a distinct cluster of PGCs is detectable anterior of the MPGZ following EdU pulse-labeling. Indeed the PGCs form independently from the stem cells of the MPGZ prior to gastrulation. Our data suggest an early PGC formation in the polychaete by preformation rather than by epigenesis.
RESUMO
Germ cells in hydrozoans arise lifelong from multipotent interstitial stem cells. To discover if a true germline-soma segregation exists in these species, we studied gametogenesis in Hydractinia echinata using in situ hybridization and immunohistochemistry for the germ cell marker Vasa. We could show that Hevas is a zygotic transcript, present in embryos from the gastrula stage onward. In the planula larva, Hevas is expressed in the interstitial stem cells located in the endoderm. During metamorphosis, Hevas-expressing cells appear in the ectoderm in the lower half of the polyp. While the Hevas transcript is not detectable in developing gametes, the protein accumulates during oogenesis. Vasa containing granules are detectable at the polar-body-forming pole after fertilization. These results suggest that, in Hydractinia, maternal Vasa protein, but not the mRNA, is a maternal constituent of a germ plasm and might be involved in the specification and maintenance of interstitial stem cells.