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her6is a zebrafish ortholog ofHes1, known for its role in maintaining neural progenitors during neural development. Here, we characterise the population-level effect of altering Her6 protein expression dynamics at the single-cell level in the embryonic zebrafish telencephalon. Using an endogenousHer6:Venusreporter and 4D single-cell tracking, we show that Her6 oscillates in neural telencephalic progenitors and that fusion of a protein destabilisation domain (PEST) to Her6:Venus alters its expression dynamics causing most cells to downregulate Her6 prematurely. However, in PEST mutants, a higher proportion of cells exhibit Her6 oscillations and while expression is reduced in most cells, some cells express Her6 at wild-type levels resulting in increased heterogeneity of Her6 expression in the population. Despite the profound differences in the single-cell Her6 dynamics, differentiation markers do not exhibit major differences early on, while an increase in differentiation is observed at later developmental stages (vglut2a, gad1andgad2). At the same time, at late stage the overall size of the telencephalon remains the same. Computational modelling that simulates changes in Her6 protein stability reveals that the increase in population Her6 expression heterogeneity is an emergent property of finely tuned Notch signalling coupling between single cells. Our study suggests that such cell coupling provides a compensation strategy whereby a normal phenotype is maintained while single-cell dynamics are abnormal, although the limit of this compensation is reached at late developmental stages. We conclude that in the neural progenitor population, cell coupling controls Her6 expression heterogeneity and in doing so, it provides phenotypic robustness when individual cells lose Her6 expression prematurely.

In recent years, record numbers of unaccompanied immigrant children have migrated to the USA, with 2019 being the highest year yet. The majority of unaccompanied children have overcome traumatic experiences and violations of their human rights in their home countries or on their journey to the USA, and/or in US detention centers. The Universal Declaration of Human Rights outlines guaranteed basic rights for every person everywhere, and the National Association of Social Workers code of ethics outlines social workers’ responsibility to challenge social injustices. Social workers have a role in advocating for the human rights of all people, including unaccompanied children. Macro social work roles—including roles such as advocacy and policy work, research, capacity building, community education, and management—are critical to addressing and ameliorating the human rights violations faced by unaccompanied immigrant children (UC). In this article, we introduce unaccompanied children as a vulnerable population, outline the human rights violations they commonly face in the USA, and offer implications and suggestions for schools of social work, social work curricula, and social work field education. We believe that effective training and field placements will better prepare the next generation of social workers and create a pipeline of knowledgeable professionals to help unaccompanied children and their families. Therefore, we highlight ways in which schools of social work, field placement agencies, field supervisors, and students can work to advance the lives of UC by offering specific examples of macro roles for interns.

Here, we study the dynamical expression of endogenously labelled Hes1, a transcriptional repressor implicated in controlling cell proliferation, to understand how cell-cycle length heterogeneity is generated in ER+ breast cancer cells. We find that Hes1 shows oscillatory expression with approximately 25h periodicity and during each cell-cycle has a variable peak in G1, a trough around G1-S transition and a less variable second peak in G2/M. Compared to other subpopulations, the cell-cycle in CD44HighCD24Low cancer stem cells is longest and most variable. Most cells divide around the peak of the Hes1 expression wave but preceding mitoses in slow dividing CD44HighCD24Low cells appear phase-shifted, resulting in a late-onset Hes1 peak in G1. The position, duration and shape of this peak, rather than the Hes1 expression levels, are good predictors of cell-cycle length. Diminishing Hes1 oscillations by enforcing sustained expression slows down the cell-cycle, impairs proliferation, abolishes the dynamic expression of p21, and increases the percentage of CD44HighCD24Low cells. Reciprocally, blocking the cell-cycle causes an elongation of Hes1 periodicity, suggesting a bidirectional interaction of the Hes1 oscillator and the cell-cycle. We propose that Hes1 oscillations are functionally important for the efficient progression of the cell-cycle and that the position of mitosis in relation to the Hes1 wave underlies cell-cycle length heterogeneity in cancer cell subpopulations.Significance statementTumours exhibit heterogeneities that are not due to mutations, including Cancer Stem Cells with different potencies. We show that the cancer stem cell state predisposed to dormancy in vivo has a highly variable and long cell-cycle. Using single-cell live-imaging for the transcriptional repressor Hes1 (a key molecule in cancer), we show a new type of circadian-like oscillatory expression of Hes1 in all cells in the population. The most potent cancer stem cells tend to divide around the trough of the Hes1 oscillatory wave, a feature predictive of a long cell-cycle. A novel concept proposed here is that the position of cell division with respect to the Hes1 wave is predictive of its prospective cell-cycle length and cancer cellular sub-state.

Ultradian oscillations of HES Transcription Factors (TFs) at the single‐cell level enable cell state transitions. However, the tissue‐level organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex vivo in the developing mouse ventral spinal cord and identify microclusters of 4–6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supra‐ultradian persistence time. We show that Notch signalling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin 2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organisation of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of single‐cell dynamics., Live imaging of HES5 expression in the ventral mouse spinal cord combined with computational modelling is used to identify and analyse spatially periodic HES5 micro‐patterns that emerge from the synthesis of single cell dynamics.

Noise is prevalent in biology and has been widely quantified using snapshot measurements. This static view obscures our understanding of dynamic noise properties and how these affect gene expression and cell state transitions. Using a CRISPR/Cas9 Zebrafish her6::Venus reporter combined with mathematical and in vivo experimentation, we explore how noise affects the protein dynamics of Her6, a basic helix‐loop‐helix transcriptional repressor. During neurogenesis, Her6 expression transitions from fluctuating to oscillatory at single‐cell level. We identify that absence of miR‐9 input generates high‐frequency noise in Her6 traces, inhibits the transition to oscillatory protein expression and prevents the downregulation of Her6. Together, these impair the upregulation of downstream targets and cells accumulate in a normally transitory state where progenitor and early differentiation markers are co‐expressed. Computational modelling and double smFISH of her6 and the early neurogenesis marker, elavl3, suggest that the change in Her6 dynamics precedes the downregulation in Her6 levels. This sheds light onto the order of events at the moment of cell state transition and how this is influenced by the dynamic properties of noise. Our results suggest that Her/Hes oscillations, facilitated by dynamic noise optimization by miR‐9, endow progenitor cells with the ability to make a cell state transition., Live imaging at single‐cell resolution shows that miR‐9 counteracts noise in oscillatory expression of transcriptional repressor Her6 to drive neural progenitor differentiation in zebrafish hindbrain.

Resumen La competencia social como capacidad para crear y mantener relaciones saludables supone comportamientos valorados positivamente por la comunidad en la que un sujeto se desenvuelve. En la escuela, la competencia social cobra una gran importancia, por lo que se estima necesario analizar las tipologías sociométricas del estudiantado en función del sexo y curso académico. En esta investigación participaron 772 estudiantes de 26 cursos diferentes, pertenecen a 8 comunas de la región de Ñuble, 420 niños (54.4 %) y 352 niñas (45.6), 250 cursan 4° año, 274 cursan 5° año y 248 cursan 6° año de primaria. El instrumento utilizado es el Cuestionario sociométrico de nominaciones entre iguales, instrumento que proporciona información sobre la niñez rechazada, ignorada, controvertida y medios preferidos en sus cursos. Los datos fueron analizados con el software SOCIOMET. Los resultados indican un mayor porcentaje de niños rechazados, ignorados y preferidos, que de niñas en estas tipologías, hecho que se reitera también por curso académico. Se puede observar, además, que los índices de amistad en los distintos cursos superan los índices de enemistad. A partir de estos resultados, se estima que pueden proyectarse intervenciones ajustadas a los requerimientos contextuales. Abstract Social competence, seen as a capacity to create and maintain healthy relationships, involves behaviors valued positively by the community in which a subject develops. At school, the social competence acquires great importance; this is the reason why analyzing the sociometric typologies of the students according to the sex and academic course is considered necessary. This research involved 772 students from 26 different classrooms; they live in eight communities in the Ñuble region. There were 420 children (54.4%) and 352 girls (45.6%); of them, 250 children are in 4th grade, 274 in 5th grade, and 248 in 6th grade of elementary school. The instrument used is the Sociometric Questionnaire of peer nominations; it provides information about children who are rejected, ignored, and controversial; it also collects information about the average of preferred children in classrooms. The data were analyzed with the SOCIOMET software. The results indicate a higher percentage of rejected, ignored, and preferred boys than of girls in these typologies; this fact is also reiterated by the academic year. Besides, the indices of friendship in the different classrooms surpass the indices of enmity. Based on these results, interventions tailored to contextual requirements can be projected. Resumo A competência social como capacidade de criar e manter relacionamentos saudáveis envolve comportamentos valorizados positivamente pela comunidade em que um sujeito se desenvolve. Na escola, a competência social adquire uma grande importância pelo que se considera necessário analisar as tipologias sociométricas dos estudantes segundo o sexo e o curso acadêmico. Esta pesquisa envolveu 772 estudantes de 26 cursos diferentes, que pertencem a 8 municípios na região de Dims, 420 meninos (54.4%) e 352 meninas (45.6%), deles 250 crianças matriculadas no 4º ano, 274 no 5º ano e 248 estão no 6º ano do ensino fundamental. O instrumento utilizado é o Questionário Sociométrico de indicações de pares; instrumento que fornece informações sobre crianças rejeitadas, ignoradas, controversas, e os meios preferidos nos seus cursos. Os dados foram analisados com o software SOCIOMET. Os resultados indicam uma porcentagem maior de meninos rejeitados, ignoradas e preferidas que as meninas, dentro dessas tipologias, fato que também é reiterado no ano letivo. Observa-se, ademais, que os índices de amizade nos diferentes cursos superam os índices de inimizade. Com base nesses resultados, estima-se que as intervenções adaptadas aos requisitos contextuais possam ser projetadas.

Ultradian oscillations of key transcription factors, such as members of the Hes family, are thought to be important in Neural Progenitor Cell (NPC) maintenance and miR-9 acts as a tuner of these oscillations in vitro. However, the existence and the role of such dynamic oscillatory expression in vivo is poorly understood. Here, we have generated a Zebrafish CRISPR knock-in Her6::venus fusion (Hes1 orthologue) to study endogenous dynamic gene expression in the embryonic hindbrain. We show that Her6 undergoes a transition from irregular, noisy, fluctuations to periodic oscillations as neurogenesis proceeds. In the absence of miR-9 input, noise in the Her6 oscillator increases and NPCs are unable to transit away from an intermediary state where they co-express progenitor and early differentiation markers. Thus, Her6 oscillations are facilitated by noise optimization mediated by miR-9 and this noise-tuning step is functionally important for cells to transition to differentiation.

Agriculture in the Altiplano and Andean Mountains is experiencing threats to sustainability mainly due to intensive cultivation of quinoa driven by international markets. This recent export-oriented production system is causing the degradation of soils and reducing productivity, therefore, agro-technological innovations are necessary to sustain cropping systems while maintaining organic quality (mostly quinoa). In this work, we searched for native Trichoderma species associated with plants from the Andean highlands to obtain an environmentally friendly and organic alternative to chemical fertilizers. We obtained different Trichoderma isolates from quinoa, potato and maize roots and soil, which were identified as Trichoderma harzianum, as well as other species. Twelve of the isolates were cultured in pairs to stimulate the production and secretion of compounds of diverse chemical nature that we called collectively ‘secondary metabolites’ (SMs). Crude extracts of SMs were used to inoculate selected crops to determine their plant growth promoting potential compared with two commercially available controls, chemical fertilizer and a bio-fertilizer. Results showed that SMs significantly promoted lettuce and radish growth and increased quinoa grain yield. Indole acetic acid was detected in all SM extracts that promoted plant growth, suggesting that this plant regulator might be responsible for the plant growth promoting activity. In conclusion, the Trichoderma-derived SMs approach appears to be a promising, simple and accessible technology for small-scale farmers in order to insure the sustainability, affordability and accessibility of food production in the Andes.

De acuerdo a la ciencia de la ocupación y la justicia ocupacional, la participación e inclusión en ocupaciones significativas, resulta fundamental para el desarrollo de las personas y de las comunidades, sin embargo, esta inclusión no siempre se ve favorecida en todos los espacios públicos. El ámbito de la educación ha desarrollado un avance importante en esta materia principalmente en sus niveles primarios, potenciando proyectos como el de integración escolar, con lo cual se ha dado espacio a alumnos en situación de discapacidad. Estos proyectos no se han incorporado a la educación superior, por lo cual los alumnos deben crear sus propias estrategias para la inclusión ya que los ambientes tanto físicos como sociales no siempre están preparados para responder a las necesidades particulares de los alumnos con distintas discapacidades y otorgar un real y equitativo acceso a la educación. Esta investigación busca obtener mayores antecedentes acerca de los procesos de inclusión y adaptación de un grupo de jóvenes con discapacidad en la Universidad de Magallanes, con el fin de aportar de manera concreta a estas políticas. Para esto, se utilizó la metodología cualitativa, considerando la experiencia de 15 estudiantes, de diversas carreras y con distinto tipo de discapacidad. Dentro del estudio, aparecen como relevantes los facilitadores vinculados al entorno, referente a ayudas técnicas, familia y red social, y por otra parte, las características personales de los estudiantes, como por ejemplo la capacidad de resiliencia que les permite sobreponerse y responder con estrategias personales a los desafíos y barreras existentes. Por lo anterior, aparecen necesidades que invitan a potenciar el cambio de paradigma de la inclusión en el ámbito educativo, que contemple un equilibrio real entre considerar los requerimientos especiales para la equidad y a su vez fomentar el trato igualitario que promueva la dignidad de las personas con discapacidad.

Immature stage VI Xenopus oocytes are arrested at the G2/M border of meiosis I until exposed to progesterone, which induces meiotic resumption through a non-genomic mechanism. One of the earliest events produced by this hormone is inhibition of the plasma membrane enzyme adenylyl cyclase (AC), with the concomitant drop in intracellular cAMP levels and reinitiation of the cell cycle. Recently Gsα and Gβγ have been shown to play an important role as positive regulators of Xenopus oocyte AC, maintaining the oocyte in the arrested state. However, a question that still remains unanswered, is how the activated state of Gsα and Gβγ is achieved in the immature oocyte, since no receptor or ligand have been found to be required. Here we provide evidence that xRic-8 can act in vitro and in vivo as a GEF for Gsα. Overexpression of xRic-8, through mRNA injection, greatly inhibits progesterone induced oocyte maturation and endogenous xRic-8 mRNA depletion, through siRNA microinjection, induces spontaneous oocyte maturation. These results suggest that xRic-8 is participating in the immature oocyte by keeping Gsα-Gβγ-AC signaling complex in an activated state and therefore maintaining G2 arrest. J. Cell. Physiol. 214: 673–680, 2008. © 2007 Wiley-Liss, Inc.

Cystic echinococcosis is the most prevalent zoonosis in Tierra del Fuego province, Argentina, with important economic, productive and public health consequences. The present work was performed to determine the ovine prevalence in Tierra del Fuego, Argentina, as well as to evaluate the quality of diagnostic systems in slaughterhouses. Moreover, genetic analyses to characterize the strain of Echinococcus granulosus involved in the region were done. The first actions to perform a diagnosis of the epidemiological situation of hydatidosis in Tierra del Fuego were done between 1976 and 1977. A canine prevalence of 80% and an ovine prevalence of 55% results were obtained. Since 1979 the control program of Hydatidosis of Tierra del Fuego was implemented. It was based on semiannual canine anthelmintic treatment with praziquantel at dose of 5mg/kg, and complemented with sanitary education and canine and ovine epidemiological surveillance. During May 1997-January 1999: 5,916 sheep coming from 20 farms of the programmatic area were evaluated. In the lamb category, hydatid cysts were not found. In the adults category, 62 infected animals were found (3.2%). The ovine prevalence was 1.1% and there was 100% of coincidence between diagnosis in the slaughterhouse, re-inspection in the laboratory and histopathological study. The marked decrease in the prevalence observed for sheep infection evidenced a destabilization of the biological cycle of the parasite. This could be explained by the application of a control program with uninterrupted systematic actions. Polymerase chain reaction-ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR-RFLP) analysis and partial sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene were used to characterize E. granulosus isolates collected from different regions of Tierra del Fuego to determine which genotypes occurred in this region. The results revealed the presence of the G1 genotype (sheep-dog strain). This is the first time that a molecular analysis was performed for the E. granulosus isolates from Tierra del Fuego.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.

Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.

G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface.

Wound healing is essential for survival. We took advantage of the Xenopus embryo, which exhibits remarkable capacities to repair wounds quickly and efficiently, to investigate the mechanisms responsible for wound healing. Previous work has shown that injury triggers a rapid calcium response, followed by the activation of Ras homolog (Rho) family guanosine triphosphatases (GTPases), which regulate the formation and contraction of an F-actin purse string around the wound margin. How these processes are coordinated following wounding remained unclear. Here we show that inositol-trisphosphate 3-kinase B (Itpkb) via its enzymatic product inositol 1,3,4,5-tetrakisphosphate (InsP 4 ) plays an essential role during wound healing by modulating the activity of Rho family GTPases and F-actin ring assembly. Furthermore, we show that Itpkb and InsP 4 modulate the speed of the calcium wave, which propagates from the site of injury into neighboring uninjured cells. Strikingly, both overexpression of itpkb and exogenous application of InsP 4 accelerate the speed of wound closure, a finding that has potential implications in our quest to find treatments that improve wound healing in patients with acute or chronic wounds.

El fallo a analizar no solo es interesante por la determinación de la conducta que los departamentos de tránsito deben exigir al momento de otorgar duplicados de las licencias de conducir, cuestión que en la práctica no se cumple, sino que además porque la Corte Suprema realiza una acertada aplicación de principios fundamentales del ordenamiento jurídico penal, como es el principio de legalidad. The following leading case in analysis is interesting not only because set the requirements that the Traffic Departments should require when granting a duplicate of a driver's licenses, but also because the Supreme Court makes a successful application of significant Criminal Law principles as the legality principle.

The molecular mechanisms that underlie the development of primitive myeloid cells in vertebrate embryos are not well understood. Here we characterize the role of cebpa during primitive myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Loss- and gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally, we use live imaging to show that cebpa-expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus, C/EPBα is the first known single factor capable of initiating an entire myelopoiesis pathway in pluripotent cells in the embryo.

Vertebrate blood formation occurs in 2 spatially and temporally distinct waves, so-called primitive and definitive hematopoiesis. Although definitive hematopoiesis has been extensively studied, the development of primitive myeloid blood has received far less attention. In Xenopus, primitive myeloid cells originate in the anterior ventral blood islands, the equivalent of the mammalian yolk sac, and migrate out to colonize the embryo. Using fluorescence time-lapse video microscopy, we recorded the migratory behavior of primitive myeloid cells from their birth. We show that these cells are the first blood cells to differentiate in the embryo and that they are efficiently recruited to embryonic wounds, well before the establishment of a functional vasculature. Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors. Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate. Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/β-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Gαq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGαq) in dorso-ventral axis embryo patterning. Overexpression of xGαq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGαq dominant-negative mutant reverted the xGαq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGαq activation, indicating that it is negatively regulating the Wnt/β-catenin pathway. J. Cell. Physiol. 214: 483–490, 2008. © 2007 Wiley-Liss, Inc.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.

Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.

G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface.

Summary Embryonic wound healing provides a perfect example of efficient recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic wound healing requires two functionally distinct actin structures: a contractile actomyosin cable and actin protrusions at the leading edge. Here, we report that the discrete formation and function of these two structures is achieved by the temporal segregation of two intracellular upstream signals and distinct downstream targets. The sequential activation of ERK and phosphoinositide 3-kinase (PI3K) signalling divides Xenopus embryonic wound healing into two phases. In the first phase, activated ERK suppresses PI3K activity, and is responsible for the activation of Rho and myosin-2, which drives actomyosin cable formation and constriction. The second phase is dominated by restored PI3K signalling, which enhances Rac and Cdc42 activity, leading to the formation of actin protrusions that drive migration and zippering. These findings reveal a new mechanism for coordinating different modes of actin-based motility in a complex tissue setting, namely embryonic wound healing.