Histology of Organogenic and Embryogenic Responses in Cotyledons of Somatic Embryos of Quercus Suber L.

In cork oak (Quercus suber L.), recurrent embryogenesis is produced in vitro through autoembryony without exogenous plant growth regulators (PGRs); secondary embryos appear on the embryo axis but seldom on cotyledons. Focusing mainly on the histological origin of neoformations, we investigated the influence of the embryo axis and exogenous PGRs on the embryogenic potential of somatic embryo cotyledons. Isolated cotyledons of somatic embryos became necrotic when cultured on PGR-free medium but gave secondary embryos when cultured on media containing benzyladenine and naphthaleneacetic acid. Cotyledons of cork oak somatic embryos are competent to give embryogenic responses. Isolated cotyledons without a petiole showed a lower percentage of embryogenic response than did those with a petiole. In petioles, somatic embryos arose from inner parenchyma tissues following a multicellular budding pattern. Joined to the embryo axis, cotyledons did not show morphogenic responses when cultured on PGR-free medium but revealed budlike and phylloid formations when cultured on medium with PGRs. The different morphogenic behavior displayed by somatic cotyledons indicates an influence of the embryo axis and indicates a relationship between organogenic and embryogenic regeneration pathways

Supernumerary molars. A review of 53 cases

Supernumerary molars are not uncommon and may be found nearly anywhere in the dental alveolar arches. A series of 36 patients that presented with 53 supernumerary molars are reviewed. They occurred more frequently in the maxilla (86.8%), had little or no clinical significance, tended to be impacted, and were not associated with the impactation of the third molar. Four cases of maxillary fifth molars are described

Supernumerary premolars. Report of 10 cases

Supernumerary premolars are 'extra' teeth morphologically belonging to the premolar group. Hyperdontia affecting premolars can be single (when only one supernumerary tooth is found), multiple (if several teeth are involved) or related to a syndrome such as cleidocranial dysplasia. Supernumerary premolars are the third most frequent group of supernumerary teeth, following mesiodens and fourth molars; the prevalence among the general population ranges from 0.09-0.64%. The present study describes 10 cases of supernumerary premolars treated in our Service of Oral and Maxillofacial Surgery of the Dental Clinic of the University of Barcelona. A late onset was demonstrated in one case, contrasting the present pantomograph with another obtained 5 years before. In only one case did the presence of a supernumerary premolar alter normal tooth eruption. Two supernumerary premolars were affected by follicular cysts

Disruption of embryonic blood-CSF barrier in chick embryos reveals the actual importance of this barrier to control E-CSF composition and homeostasis in early brain development

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube. After closure of the anterior neuropore, the brain wall forms a physiologically sealed cavity that encloses embryonic cerebrospinal fluid (E-CSF), a complex and protein-rich fluid that is initially composed of trapped amniotic fluid. E-CSF has several crucial roles in brain anlagen development. Recently, we reported the presence of transient blood-CSF barrier located in the brain stem lateral to the ventral midline, at the mesencephalon and prosencephalon level, in chick and rat embryos by transporting proteins, water, ions and glucose in a selective manner via transcellular routes. To test the actual relevance of the control of E-CSF composition and homeostasis on early brain development by this embryonic blood-CSF barrier, we block the activity of this barrier by treating the embryos with 6-aminonicotinamide gliotoxin (6-AN). We demonstrate that 6-AN treatment in chick embryos blocks protein transport across the embryonic blood-CSF barrier, and that the disruption of the barrier properties is due to the cease transcellular caveolae transport, as detected by CAV-1 expression cease. We also show that the lack of protein transport across the embryonic blood-CSF barrier influences neuroepithelial cell survival, proliferation and neurogenesis, as monitored by neurepithelial progenitor cells survival, proliferation and neurogenesis. The blockage of embryonic blood-CSF transport also disrupts water influx to the E-CSF, as revealed by an abnormal increase in brain anlagen volume. These experiments contribute to delineate the actual extent of this blood-CSF embryonic barrier controlling E-CSF composition and homeostasis and the actual important of this control for early brain development, as well as to elucidate the mechanism by which proteins and water are transported thought transcellular routes across the neuroectoderm, reinforcing the crucial role of E-CSF for brain development.

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

Multiple supernumerary teeth not associated with complex syndromes: a retrospective study

Objectives: To determine the epidemiology and describe the clinical and radiographic characteristics, the type of treatment, and the possible delayed appearance of new supernumerary teeth in patients with non-syndromic multiple hyperdontia. Patients and Methods: We conducted a small retrospective observational study of 8 patients diagnosed with nonsyndromic multiple hyperdontia. Multiple hyperdontia not associated to complex syndromes was defined as apparently generally healthy patients with one or more supernumerary teeth in two or more areas. Results: The average patient age was 16.23 years; males predominated (3:1). Multiple hyperodontia with a minimum of 2 and a maximum of 9 supernumerary teeth was found (total: 34 mean: 4.25). The most frequent location was the upper jaw (76.47%). Eumorphic teeth were seen at lower premolar level, while the rest were all heteromorphic. There was altered eruption of the contiguous teeth of 4 of the impacted supernumerary teeth all the rest being asymptomatic. Extraction was the treatment in all patients, and in one of them the delayed appearance of 4 supernumerary teeth was detected. Conclusions: Multiple hyperodontia rarely occurs without being associated with complex syndromes. Prophylactic surgical removal of the supernumerary teeth is generally the treatment of choice.

Retrospective study of 145 supernumerary teeth

Objective: The goal of the present retrospective study is to describe the distribution of the supernumerary teeth in a population of patients that have been attended at the Public Clinic of the Department of Oral Surgery. Background: Supernumerary teeth and multiple hyperdontia are usually associated with different syndromes, such as Gardner syndrome, or with facial fissures; however, they can appear in patients without any pathology. Their prevalence oscillates to 0.5-3.8% in patients with permanent teeth and to 0.35-0.6% in patients with primary teeth. Patients and Methods: A total of 36,057 clinical histories of patients that were admitted at the clinic between September of 1991 and March of 2003 were revised. The following data were extrapolated: age, sex, number of extracted supernumerary teeth, localization, morphology and type of supernumerary teeth. Consequently, 102 patients were included into the present study. Results: Of the 147 supernumerary teeth identified in the oral cavities of patients 145 were extracted. The most frequent supernumerary teeth identified were mesiodens (46.9%), followed by premolars (24.1%) and fourth molars or distal molars (18%). As for location, 74.5% of the supernumerary teeth were found in the superior maxillary bone and 46.9% of the supernumerary teeth were present in the palatine/lingual area. Heteromorphology was found in two thirds of the supernumerary teeth, with conical shape being the most frequent. Finally, 29.7% of the supernumerary teeth had occlusion with permanent teeth, and mesiodens were the predominating type of supernumerary teeth that showed this feature. Conclusions: Mesiodens very frequently cause retention of permanent incisors, which erupt spontaneously after the extraction of supernumerary teeth, if there is sufficient space in the dental arch and if they conserve the eruptive force. Generally, supernumerary premolars are eumorphic and are casually discovered during radiological exam, if not producing any symptomology.

Maxillary dentigerous cyst and supernumerary tooth. Is it a frequent association?

Dentigerous cysts, also known as follicular cysts, are a relatively common pathology in our field. They are associated with unerupted or semi-erupted teeth and are usually not related to supernumerary teeth. OBJECTIVE: To describe a dentigerous cyst case associated to a supernumerary tooth. CASE-REPORT: A large-sized dentigerous cyst is described, associated with a supernumerary tooth, affecting the whole maxillary anterior area. Appropriate treatment consists of performing root canals and a Partsch II procedure with a cystectomy, extracting the unerupted teeth, carrying out an apicoectomy and retro-filling the affected teeth. The defect is filled with a bone xenograft. Possible therapeutic alternatives and the connection between the dentigerous cysts and supernumerary teeth are considered in the discussion.

Caracterització estructural i funcional del gen de la miogenia en l’orada (Sparus aurata )

Report for the scientific sojourn at the University of Maryland Biotechnology Institute from February to August 2007. Myogenesis of skeletal muscles in vertebrates is controlled by extracellular signalling molecules together with intracellular transcription factors. Among the transcriptional factors, the members of the myogenic regulatory family play important roles regulating skeletal muscle development and growth. To characterize the gene structure and expression of fish myogenin, we have isolated the myogenin genomic gene and cDNA from gilthead seabream (Sparus aurata) and analyzed the genomic structure, pattern of expression and the regulation of musclespecific expression. Sequence analysis revealed that the seabream myogenin shares a similar gene structure with other fish myogenins, with three exons, two introns and the highly conserved bHLH domain. Expression studies demonstrated that myogenin is expressed in both slow and fast muscles as well as in muscle cells in primary culture. In situ hybridization showed that myogenin was specifically expressed in developing somites of seabream embryos. Promoter activity analysis demonstrated that the myogenin promoter could drive green fluorescence protein expression in muscle cells of zebrafish embryos, as well as in myofibers of adult zebrafish and juvenile seabream.

Análisis comparativo de tres técnicas de derivación de células madre

Las células madre embrionarias (Embryonic Stem Cells; ESC) son células pluripotentes que presentan la capacidad de dividirse indefinidamente a la vez que mantienen la habilidad para diferenciarse a cualquier tipo celular. Aunque de manera rutinaria se derivan a partir de la masa celular interna de embriones en estadio de blastocisto, también pueden derivarse a partir de embriones en estadios precompactacionales y de embriones reconstruidos por procesos de transferencia nuclear. Debido a que durante el desarrollo embrionario temprano, momento en el que se derivan las ESC, tienen lugar profundos cambios de metilación en el genoma, tanto la derivación como el cultivo se consagran como técnicas que pueden alterar los patrones de metilación en genes regulados por impronta genómica. Con el objetivo de analizar la estabilidad epigenética de embriones preimplantacionales y ESC murinas, en este trabajo se ha optimizado un protocolo de anàlisis de los niveles de metilación mediante pirosecuenciación. Para ello se han seleccionado tres genes regulados por impronta genómica (H19/Igf2, Snrpn and Peg3), dos genes relacionados con el mantenimiento de pluripotencia en ESC (Oct4, Nanog y Sox2) y dos genes marcadores de diferenciación temprana (Cdx2 y Gata6). Nuestros resultados muestran que algunos grupos de embriones preimplantacionales presentan una hipo e hipermetilación en las regiones diferencialmente metiladas (Differentially Methylated Regions, DMRs) de los genes Snrpn y Peg3. Además, la línea de ESC analizada presentó anomalías en los tres genes regulados por impronta genómica. No obstante, el hecho de que esta línea fuera inestable a nivel cariotípico no permite establecer una relación entre el cultivo in vitro o la técnica de derivación y la inestabilidad epigenética demostrada. Por todo esto, parece pertinente analizar tanto la integridad epigenética como la estabilidad cromosómica de ESC antes de proceder a realizar ensayos clínicos en humanos.

Avaluació de la teratogènia in vitro mitjançant el cultiu d’embrions sencers (WEC)

Projecte de recerca elaborat a partir d’una estada a la Charité - Universitätsmedizin Berlin, Alemanya, entre novembre i desembre del 2007. En aquest treball es presenta el protocol a seguir per a dur a terme el cultiu d’embrions sencers in vitro (Whole Embryo Culture, WEC). Amb aquest protocol es pretén implementar la tècnica del WEC en el laboratori de la Unitat de Toxicologia de la Facultat de Farmàca (UB), seguint la metodologia apresa durant l’estada i deixant per escrit tots els passos seguits i el material i la metodologia concreta de cadascun d’ells. En el WEC es cultiven embrions de rata de 9.5 dies durant 48h en ampolles rotatòries en un medi líquid i amb una fase gasosa controlats. Durant el cultiu, tenen lloc dos processos principals: el plegament de l’embrió i l’organogènesi. Els embrions durant els dos dies que dura el cultiu es pleguen en els plans transversal i sagital, passant d’un embrió pla a un altre de cilíndric en forma de “C”. En aquest període, a més, es produeixen importants processos d’organogènesi com la neurulació, la formació de la cresta neural, dels somites, dels vasos sanguinis - el cor inclòs- i de la sang. Es comencen a formar la placoda nasal, la vesícula oftàlmica, la vesícula òtica, les extremitats superiors i inferiors i la cua. En la memòria adjunta es descriuen amb detall els processos d'aparellament dels animals, preparació del material i del medi de cultiu, el procés d'aïllament del embrions en el dia 9.5, les condicions de cultiu i l'avaluació dels embrions en el dia 11.5. Finalment es presenten resultats d'embrions en situació control amb un correcte desenvolupament i es mostra com, al final de l'estada, es va aconseguir el cultiu d’embrions control amb un desenvolupament correcte i estadísticament sense diferències respecte als diferents paràmetres mesurats en comparació amb els embrions control de la Charité-Universitätsmedizin de Berlin.

Generation of cardiomyocytes from new human embrionic stem cell lines derived from poor-quanlity blastocysts

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently, hES cell lines are derived from surplus embryos from assisted reproduction cycles, independent of their quality or morphology. Here, we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore, we show that the self-renewal, pluripotency, and differentiation ability of hES cell lines derived from either source are comparable. Finally, we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine.

Constitutive expression of FGF4 disrupts the development of the eye and the anterior CNS during mouse embryogenesi, but does not influence the expressionof shh in these areas.

The biological consequences of constitutive fibroblast growth factor-4 (fgf4) expression have been analysed during anterior CNS development of mouse chimeric embryos. Severe mutant embryos exhibit exencephaly, absence of eye development and anomalous differentiation of neuropithelium. These embryos also show ectopic limb buds resembling the early phases of limb development. Because our results show that anterior CNS in those chimeric embrios does not express shh ectopically, we suggest that malformations may be due to interference between the ectopic expression of fgf4 in the cephalic area and the receptors for the members of the FGF family that regulate brain and eye development, namely fgf8. If this is correct, the results indirectly suport the crucial role of fgf8 in patterning the anterior CNS.

Effects of Different Salinities on Juvenile Growth of Gammarus aequicauda (Malacostraca: Amphipoda)

Gammarus aequicauda is a euryhaline amphipod that is a common inhabitant of brackish environments of theMediterranean Sea. In the Ebro delta, the population density of G. aequicauda is highly variable throughout the year. The main objective of this study is to investigate the effect of salinity on the growth of G. aequicauda juveniles. G. aequicauda embryos and juveniles can survive and grow in the laboratory between 2 psu and 40 psu salinity, depending on the previous acclimation period for the reproductive individuals. Adults acclimated at 34 psu produced embryos and juveniles that survived and developed at salinities between 9 psu and 40 psu; adults acclimated at 9 psu produced embryos and juveniles that could develop in oligohaline conditions. The lower growth rate values were 10.9 μmd−1 and 13.5 μmd−1 at 40 psu and 2 psu, respectively, with the higher values of 18.0 μmd−1 and 18.5 μmd−1 at 19 and 34 psu, respectively.

Genome-wide chromatin occupancy reveals a role for ASH2 in transcriptional pausing

While it is widely acknowledged that the ubiquitin-proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.