RESUMEN
Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Lateralidad Funcional , Animales , Dominios Proteicos , ARN Mensajero/genética , Ribonucleoproteínas/genéticaRESUMEN
For left-right symmetry breaking in the mouse embryo, the basal body must become positioned at the posterior side of node cells, but the precise mechanism for this has remained unknown. Here, we examined the role of microtubules (MTs) and actomyosin in this basal body positioning. Exposure of mouse embryos to agents that stabilize or destabilize MTs or F-actin impaired such positioning. Active myosin II was detected at the anterior side of node cells before the posterior shift of the basal body, and this asymmetric activation was lost in Prickle and dachsous mutant embryos. The organization of basal-body associated MTs (baMTs) was asymmetric between the anterior and posterior sides of node cells, with anterior baMTs extending horizontally and posterior baMTs extending vertically. This asymmetry became evident after polarization of the PCP core protein Vangl1 and before the posterior positioning of the basal body, and it also required the PCP core proteins Prickle and dachsous. Our results suggest that the asymmetry in baMT organization may play a role in correct positioning of the basal body for left-right symmetry breaking.
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Cuerpos Basales , Polaridad Celular , Actinas/metabolismo , Animales , Polaridad Celular/fisiología , Cilios/metabolismo , Ratones , Microtúbulos/metabolismoRESUMEN
Gse1 is a component of the CoREST complex that acts as an H3K4 and H3K9 demethylase and regulates gene expression. Here, we examined the expression and role of Gse1 in mouse development. Gse1 is expressed in male and female germ cells and plays both maternal and zygotic roles. Thus, maternal deletion of Gse1 results in a high incidence of prenatal death, and zygotic deletion leads to embryonic lethality from embryonic day 12.5 (E12.5) and perinatal death. Gse1 is expressed in the junctional zone and the labyrinth of the developing placenta. Gse1 mutant (Gse1Δex3/Δex3) placenta begins to exhibit histological defects from E14.5, being deficient in MCT4+ syncytiotrophoblast II. The number of various cell types was largely maintained in the mutant placenta at E10.5, but several genes were upregulated in giant trophoblasts at E10.5. Placenta-specific deletion of Gse1 with Tat-Cre suggested that defects in Gse1Δex3/Δex3 embryos are due to placental function deficiency. These results suggest that Gse1 is required for placental development in mice, and in turn, is essential for embryonic development.
Asunto(s)
Placenta , Placentación , Ratones , Embarazo , Femenino , Animales , Masculino , Desarrollo Embrionario/genética , TrofoblastosRESUMEN
Maternal factors present in oocytes and surrounding granulosa cells influence early development of embryos. In this study, we searched for epigenetic regulators that are expressed in oocytes and/or granulosa cells. Some of the 120 epigenetic regulators examined were expressed specifically in oocytes and/or granulosa cells. When their expression was examined in young versus aged oocytes or granulosa cells, many were significantly up- or downregulated in aged cells. The maternal role of six genes in development was investigated by generating oocyte-specific knock-out (MKO) mice. Two genes (Mllt10, Kdm2b) did not show maternal effects on later development, whereas maternal effects were evident for Kdm6a, Kdm4a, Prdm3, and Prdm16 for MKO female mice. Offspring from Kdm6a MKO mice underwent perinatal lethality at a higher rate. Pups derived from Prdm3;Prdm16 double MKO showed a higher incidence of postnatal death. Finally, embryos derived from Kdm4a MKO mice showed early developmental defects as early as the peri-implantation stage. These results suggest that many of maternal epigenetic regulators undergo differential expression upon aging. Some, such as Kdm4a, Kdm6a, Prdm3, and Prdm16, have maternal role in later embryonic or postnatal development.
Asunto(s)
Oocitos , Factores de Transcripción , Embarazo , Femenino , Animales , Ratones , Oocitos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Epigénesis Genética , Desarrollo Embrionario/genéticaRESUMEN
The processes of development leading up to gastrulation have been markedly altered during the evolution of amniotes, and it is uncertain how the mechanisms of axis formation are conserved and diverged between mouse and chick embryos. To assess the conservation and divergence of these mechanisms, this study examined gene expression patterns during the axis formation process in Chinese soft-shell turtle and Madagascar ground gecko preovipositional embryos. The data suggest that NODAL signaling, similarly to avian embryos but in contrast to eutherian embryos, does not have a role in epiblast and hypoblast development in reptilian embryos. The posterior marginal epiblast (PME) is the initial molecular landmark of axis formation in reptilian embryos prior to primitive plate development. Ontogenetically, PME may be the precursor of the primitive plate, and phylogenetically, Koller's sickle and posterior marginal zone in avian development may have been derived from the PME. Most of the genes expressed in the mouse anterior visceral endoderm (AVE genes), especially signaling antagonist genes, are not expressed in the hypoblast of turtle and gecko embryos, though they are expressed in the avian hypoblast. This study proposes that AVE gene expression in the hypoblast and the visceral endoderm could have been independently established in avian and eutherian lineages, similar to the primitive streak that has been independently acquired in these lineages.
Asunto(s)
Tipificación del Cuerpo/fisiología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Lagartos/embriología , Tortugas/embriología , Animales , Blastodermo/fisiología , Tipificación del Cuerpo/genética , Endodermo/metabolismo , Gastrulación/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Lagartos/genética , Lagartos/metabolismo , Proteína Nodal/fisiología , Filogenia , Línea Primitiva/metabolismo , Especificidad de la Especie , Factores de Transcripción/fisiología , Tortugas/genética , Tortugas/metabolismoRESUMEN
BACKGROUND: Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior-posterior (A-P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A-P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. RESULTS: All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2-positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp-positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A-P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. CONCLUSIONS: Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents.
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Ectodermo/fisiología , Desarrollo Embrionario/fisiología , Endodermo/fisiología , Regulación del Desarrollo de la Expresión Génica , Animales , Tipificación del Cuerpo/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Nodal/genética , Conejos , PorcinosRESUMEN
BACKGROUND: Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short-tailed opossum, a metatherian. RESULTS: Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post-coitus. CONCLUSIONS: Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176-1188, 2016. © 2016 Wiley Periodicals, Inc.
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Monodelphis/embriología , Monodelphis/metabolismo , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Regulación del Desarrollo de la Expresión Génica , Monodelphis/clasificación , Proteína Nodal/genética , Proteína Nodal/metabolismo , FilogeniaRESUMEN
BACKGROUND: RNA-seq enables gene expression profiling in selected spatiotemporal windows and yields massive sequence information with relatively low cost and time investment, even for non-model species. However, there remains a large room for optimizing its workflow, in order to take full advantage of continuously developing sequencing capacity. METHOD: Transcriptome sequencing for three embryonic stages of Madagascar ground gecko (Paroedura picta) was performed with the Illumina platform. The output reads were assembled de novo for reconstructing transcript sequences. In order to evaluate the completeness of transcriptome assemblies, we prepared a reference gene set consisting of vertebrate one-to-one orthologs. RESULT: To take advantage of increased read length of >150 nt, we demonstrated shortened RNA fragmentation time, which resulted in a dramatic shift of insert size distribution. To evaluate products of multiple de novo assembly runs incorporating reads with different RNA sources, read lengths, and insert sizes, we introduce a new reference gene set, core vertebrate genes (CVG), consisting of 233 genes that are shared as one-to-one orthologs by all vertebrate genomes examined (29 species)., The completeness assessment performed by the computational pipelines CEGMA and BUSCO referring to CVG, demonstrated higher accuracy and resolution than with the gene set previously established for this purpose. As a result of the assessment with CVG, we have derived the most comprehensive transcript sequence set of the Madagascar ground gecko by means of assembling individual libraries followed by clustering the assembled sequences based on their overall similarities. CONCLUSION: Our results provide several insights into optimizing de novo RNA-seq workflow, including the coordination between library insert size and read length, which manifested in improved connectivity of assemblies. The approach and assembly assessment with CVG demonstrated here would be applicable to transcriptome analysis of other species as well as whole genome analyses.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN , Animales , Benchmarking , Perfilación de la Expresión Génica/normas , Especificidad de Órganos , Estándares de Referencia , Reptiles/embriología , Reptiles/genéticaRESUMEN
Cardiovascular system develops from the lateral plate mesoderm. Its three primary cell lineages (hematopoietic, endothelial, and muscular) are specified by the sequential actions of conserved transcriptional factors. ETV2, a master regulator of mammalian hemangioblast development, however, is absent in the chicken genome and acts downstream of NPAS4L in zebrafish. Here, we investigated the epistatic relationship between NPAS4L and ETV2 in avian hemangioblast development. We showed that ETV2 is deleted in all 363 avian genomes analyzed. Mouse ETV2 induced LMO2, but not NPAS4L or SCL, expression in chicken mesoderm. Squamate (lizards, geckos, and snakes) genomes contain both NPAS4L and ETV2 In Madagascar ground gecko, both genes were expressed in developing hemangioblasts. Gecko ETV2 induced only LMO2 in chicken mesoderm. We propose that both NPAS4L and ETV2 were present in ancestral amniote, with ETV2 acting downstream of NPAS4L in endothelial lineage specification. ETV2 may have acted as a pioneer factor by promoting chromatin accessibility of endothelial-specific genes and, in parallel with NPAS4L loss in ancestral mammals, has gained similar function in regulating blood-specific genes.
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Células Madre Hematopoyéticas , Pez Cebra , Animales , Ratones , Diferenciación Celular/genética , Células Madre Hematopoyéticas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Aves , Mamíferos/metabolismoRESUMEN
We have analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer we identified immediately 3' downstream of the last coding exon is well conserved among tetrapods and unexpectedly directed all the Emx2 expression in forebrain: caudal forebrain primordium at E8.5, dorsal telencephalon at E9.5-E10.5 and the cortical ventricular zone after E12.5. Otx, Tcf, Smad and two unknown transcription factor binding sites were essential to all these activities. The mutant that lacked this enhancer demonstrated that Emx2 expression under the enhancer is solely responsible for diencephalon development. However, in telencephalon, the FB enhancer did not have activities in cortical hem or Cajal-Retzius cells, nor was its activity in the cortex graded. Emx2 expression was greatly reduced, but persisted in the telencephalon of the enhancer mutant, indicating that there exists another enhancer for Emx2 expression unique to mammalian telencephalon.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas de Homeodominio/genética , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Factores de Transcripción/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Secuencia Conservada , ADN/genética , ADN/metabolismo , Cartilla de ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Factores de Transcripción Otx/metabolismo , Fenotipo , Embarazo , Homología de Secuencia de Ácido Nucleico , Proteínas Smad/metabolismo , Especificidad de la Especie , Factores de Transcripción TCF/metabolismo , Telencéfalo/embriología , Telencéfalo/metabolismo , Factores de Transcripción/deficiencia , Xenopus/genéticaRESUMEN
Immotile cilia at the ventral node of mouse embryos are required for sensing leftward fluid flow that breaks left-right symmetry of the body. However, the flow-sensing mechanism has long remained elusive. In this work, we show that immotile cilia at the node undergo asymmetric deformation along the dorsoventral axis in response to the flow. Application of mechanical stimuli to immotile cilia by optical tweezers induced calcium ion transients and degradation of Dand5 messenger RNA (mRNA) in the targeted cells. The Pkd2 channel protein was preferentially localized to the dorsal side of immotile cilia, and calcium ion transients were preferentially induced by mechanical stimuli directed toward the ventral side. Our results uncover the biophysical mechanism by which immotile cilia at the node sense the direction of fluid flow.
Asunto(s)
Señalización del Calcio , Calcio , Cilios , Mecanotransducción Celular , Animales , Ratones , Calcio/metabolismo , Cilios/fisiología , Embrión de MamíferosRESUMEN
The epithalamus of zebrafish shows morphological and molecular left-right (L-R) asymmetry, but such asymmetry is not apparent in tetrapods. To provide further insight into the evolutionary diversity of brain L-R asymmetry, we have now examined the developing brains of reptile embryos for expression of Nodal, Lefty, and Pitx2. Two turtle species, the Chinese softshell turtle and the red-eared slider turtle, showed left-sided expression of these three genes in the developing forebrain, with this expression occurring after Nodal expression at the lateral plate and the L-R organizer has disappeared. Nodal activity, as revealed by the detection of phosphorylated Smad2/3, was also apparent in the neural epithelium on the left side in both turtle species. In the Chinese softshell turtle, the habenula did not show apparent asymmetry in size and the parapineal organ was absent, but the expression of Kctd12 in the habenula showed a small yet reproducible asymmetry. In contrast to the turtles, L-R asymmetric expression of Nodal, Lefty, Pitx2, or Kctd12 was not detected in the developing brain of the Madagascar ground gecko. The transcriptional enhancer (ASE) responsible for the asymmetric expression of Nodal, Lefty, and Pitx2 was conserved among reptiles, including the Chinese softshell turtle and Madagascar ground gecko. Our findings suggest that Nodal, Lefty, and Pitx2 have the potential to be asymmetrically expressed in the developing brain of vertebrates, but that their expression varies even among reptiles.
RESUMEN
Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) and examined the TKO (Otx1(-/-)Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Factores de Transcripción Otx/metabolismo , Animales , Secuencia de Bases , Tipificación del Cuerpo , Cartilla de ADN/genética , Diencéfalo/embriología , Diencéfalo/metabolismo , Elementos de Facilitación Genéticos , Femenino , Regulación del Desarrollo de la Expresión Génica , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Metencéfalo/embriología , Metencéfalo/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Factores de Transcripción Otx/deficiencia , Factores de Transcripción Otx/genética , EmbarazoRESUMEN
Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3' untranslated region (3'-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3'-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3'-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.
Asunto(s)
Embrión de Mamíferos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores CCR4/metabolismo , Regiones no Traducidas 3' , Animales , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Noqueados , Proteínas de Unión al ARN/genética , Receptores CCR4/genética , Canales Catiónicos TRPP/metabolismo , Factores de TranscripciónRESUMEN
To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.
Asunto(s)
Tipificación del Cuerpo , Evolución Molecular , Cabeza/embriología , Factores de Transcripción Otx/genética , Homología de Secuencia de Ácido Nucleico , Vertebrados/embriología , Animales , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Factores de Transcripción Otx/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59 cluster rafts and GM1 cluster rafts were stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK and continually recruited Lyn and H-Ras right beneath them in the inner leaflet with dwell lifetimes <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the nonraftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Because GM1 cluster rafts recruited Lyn and H-Ras as efficiently as CD59 cluster rafts, and because the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft-lipid interactions and thus serve as a key signal transduction platform.
Asunto(s)
Antígenos CD59/metabolismo , Gangliósido G(M1)/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Imagen Individual de Molécula , Familia-src Quinasas/metabolismo , Antígenos CD59/genética , Gangliósido G(M1)/genética , Células HeLa , Humanos , Microdominios de Membrana/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Familia-src Quinasas/genéticaRESUMEN
Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.
Asunto(s)
Tipificación del Cuerpo , Cilios , Animales , Embrión de Pollo , Madagascar , Ratones , Reptiles , Pez CebraRESUMEN
Overlapping of genes, especially in an anti-parallel fashion, is quite rare in eukaryotic genomes. We have found a rare instance of exon overlapping involving CHRNE and MINK gene loci on chromosome 17 in humans. CHRNE codes for the epsilon subunit of the nicotinic acetylcholine receptor (AChRepsilon) whereas MINK encodes a serine/threonine kinase belonging to the GCK family. To elucidate the evolutionary trail of this gene overlapping event, we examined the genomes of a number of primates and found that mutations in the polyadenylation signal of the CHRNE gene in early hominoids led to the overlap. Upon extending this analysis to genomes of other orders of placental mammals, we observed that the overlapping occurred at least three times independently during the course of mammalian evolution. Because CHRNE and MINK are differentially expressed, the potentially hazardous mutations responsible for the exon overlap seem to have escaped evolutionary pressures by differential temporo-spatial expression of the two genes.
Asunto(s)
Evolución Molecular , Genes Sobrepuestos/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores Nicotínicos/genética , Animales , Secuencia de Bases , Bovinos , Cromosomas Humanos Par 17/genética , ADN/química , ADN/genética , Perros , Genoma , Genoma Humano , Caballos , Humanos , Ratones , Ratones Endogámicos C57BL , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Filogenia , Poli A/genética , Primates , Conejos , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Porcinos , BallenasRESUMEN
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be shed from the cell membrane by GPI cleavage. In this study, we report a novel GPI-processing enzyme, termed post-glycosylphosphatidylinositol attachment to proteins 6 (PGAP6), which is a GPI-specific phospholipase A2 mainly localized at the cell surface. CRIPTO, a GPI-AP, which plays critical roles in early embryonic development by acting as a Nodal coreceptor, is a highly sensitive substrate of PGAP6, whereas CRYPTIC, a close homologue of CRIPTO, is not sensitive. CRIPTO processed by PGAP6 was released as a lysophosphatidylinositol-bearing form, which is further cleaved by phospholipase D. CRIPTO shed by PGAP6 was active as a coreceptor in Nodal signaling, whereas cell-associated CRIPTO activity was reduced when PGAP6 was expressed. Homozygous Pgap6 knockout mice showed defects in early embryonic development, particularly in the formation of the anterior-posterior axis, which are common features with Cripto knockout embryos. These results suggest PGAP6 plays a critical role in Nodal signaling modulation through CRIPTO shedding.
Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Nodal/metabolismo , Fosfolipasas A2/metabolismo , Animales , Tipificación del Cuerpo , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Noqueados , Modelos Biológicos , Mutación/genética , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas/genética , Transducción de SeñalRESUMEN
The p21-activated kinase (PAK) family of protein kinases has recently attracted considerable attention as an effector of Rho family of small G proteins and as an upstream regulator of MAPK signalling pathways during cellular events such as re-arrangement of the cytoskeleton and apoptosis. We have cloned a novel human PAK family kinase that has been designated as PAK5. PAK5 contains a CDC42/Rac1 interactive binding (CRIB) motif at the N-terminus and a Ste20-like kinase domain at the C-terminus. PAK5 is structurally most related to PAK4 and PAK6 to make up the PAK-II subfamily. We have shown that PAK5 preferentially binds to CDC42 in the presence of GTP and that CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5 activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly expressed in brain as revealed by Northern blot and in situ hybridization. The expression pattern of PAK5 is distinct from that of PAK4 and PAK6, suggesting a functional division among PAK-II subfamily kinases based on differential tissue distribution.