Gastrulation, literally “formation of the gut” is ultimately an inadequate term to describe one of the most dynamic periods during vertebrate developmental biology. During gastrulation coordinated cell movements reshape the non-descript blastula into the structured gastrula and simultaneously specify the three germ layers: endoderm, mesoderm and ectoderm. The morphogenetic movements of gastrulation are highly conserved between species, but the links between their genetic and biomechanical regulation are poorly understood. The zebrafish embryo – externally hatched, optically clear and amenable to genetic manipulation – is an ideal vertebrate model in which to study both morphogenetic movements and their genetic control. This thesis provides a detailed analysis of the zebrafish Mix-type homeobox transcription factor, Mtx2, both in terms of its role in gastrulation and the molecular mechanisms regulated by Mtx2. This approach involved detailed examination of the Mtx2 loss-of-function phenotype and, subsequently, use of this phenotype as the basis for a microarray screen to identify and investigate Mtx2-dependent genes. One specific Mtx2-dependent gene, katanin-like 1 was investigated further by loss-of-function studies. Prior to this study the mtx2 gene was identified by homology, within its homeodomain, to other Mix-family transcription factors, but both its function and transcriptional targets remained unknown. Using a morpholino knockdown approach, this thesis demonstrates that Mtx2 is essential for vegetal movement (epiboly), but not specification, of the embryonic germ layers and extra-embryonic tissues during zebrafish gastrulation. The recruitment of filamentous actin (F-actin) to a punctate band at the blastoderm margin, was previously shown to be responsible for progression of epiboly. However, formation of this structure is demonstrated to be Mtx2-dependent. Microarray expression profiling of the Mtx2 loss-of-function phenotype was performed to screen for novel genes with roles in gastrulation. This approach identified Mtx2-dependent genes with roles in cytoskeletal dynamics, cell-cell adhesion and endocytosis and vesicular trafficking – processes known to be involved in morphogenetic movements. Many Mtx2-dependent genes are co-expressed with mtx2 in the extra-embryonic yolk syncytial layer (YSL), the teleost functional equivalent of mammalian visceral endoderm. The subset of Mtx2-dependent genes co-expressed with mtx2 and that contain Mtx2-binding sites within their 2kb proximal promoter represent the genes with the greatest likelihood of being direct Mtx2 transcriptional targets. A novel homologue of the microtubule severing protein Katanin, known as katanin-like 1 (katnal1) met all these conditions. Morpholino knockdown of Katnal1 demonstrates that like Mtx2, Katnal1 is essential for gastrulation in zebrafish. A cloned Katnal1mCherry fusion construct was observed to associate with microtubules, and demonstrated bi-directional trafficking around transfected mammalian cells. Analysis of the microtubule network in wild-type and morpholino injected zebrafish embryos demonstrated that remodelling of the extensive microtubule network found in the YSL and yolk cytoplasmic layer (YCL) is Katnal1-dependent. Nuclear division within the YSL and F-actin recruitment to the blastoderm margin are also Katnal1-dependent. This thesis therefore demonstrates, for the first time directly, the multiple, specific roles played by the microtubule network of the YSL/YCL. Katnal1 is highly conserved from Drosophila to mammals and is dynamically expressed during mouse gastrulation. The Mtx2 binding motif in the katnal1 2kb proximal promoter can be bound by both Mtx2 and its putative mouse homologue Mixl1. This suggests that katnal1 may also be a direct target of Mtx2. At the technical level, these results demonstrate the validity of screening for novel developmentally important genes using a zebrafish microarray-based approach, the potential of such an approach to, ab initio, identify a candidate list of transcription factor targets and confirm the utility of the zebrafish as a developmental model. At the biological level, this work collectively suggests that Mtx2 is a central regulator of the morphogenetic movement of epiboly and that Katnal1-dependent microtubule remodelling drives multiple aspects of gastrulation, potentially from Drosophila through to humans.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 5 uppsatser.

Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit, that are expressed on the surface of all metazoan cells. These bidirectional signaling molecules are involved in many well-known aspects of cell function, although the role of integrins in early embryonic development remains a mystery. The purpose of this study was to characterize S. purpuratus integrins and determine if they are necessary for early embryonic development. Full length cDNA sequences for four incomplete gene predictions, αC, αD, αF, and βD, were determined by amplifying overlapping fragments and sequencing EST clones. Each cDNA has a single open reading frame predicting a protein with canonical integrin features. QPCR results show αC, αD, and βD are expressed in the embryo at relatively constant levels during the first 96 hours of development. αF is expressed in blastulae, during morphogenesis and tissue differentiation, at up to 35 times the levels of mRNA in the egg. Using a morpholino antisense oligonucleotide to block translation of αC results in a higher than normal mortality rate (57.1%) by 24 hours of development and 36.7% of embryos during this period have defects in aspects of cell division. These results indicate that αC is an essential gene for early development and that it may function in coordination of mitosis and cytokinesis. The expression of multiple subunits and the demonstration that αC has an essential role suggests that there are several non-overlapping functions for integrins in early embryonic development.

Aujourd’hui, la durabilité du modèle de développement de l’aquaculture est de plus en plus questionnée et une des solutions proposées consisterait à diversifier la production piscicole via la domestication de nouvelles espèces comme la perche commune, Perca fluviatilis, une espèce d’eau douce tempérée très intéressante pour la diversification de l’aquaculture continentale européenne. De nombreux aspects de la biologie de sa reproduction sont connus cependant, peu d’informations sont disponibles sur son développement. Or, des défauts de développement précoce, dont les causes sont encore mal définies, impactent actuellement la qualité de la production piscicole. C’est dans ce contexte que cette thèse vise à caractériser les succès et défauts de développement embryonnaire chez la perche commune à travers trois axes principaux : (i) déterminer une table de référence de l’embryogenèse normale permettant (ii) définir les défauts de développement tels que les malformations dans des conditions d’élevage différentes et (iii) identifier les liens entre différents paramètres de développement embryonnaire afin de déterminer des profils de développement variables. La première partie de ce travail a permis d’identifier la séquence précise de l’ontogenèse normale de cette espèce à travers la définition d’une table de développement embryonnaire alternative et flexible pour des espèces non-modèles, facilitant les comparaisons intra- et inter-espèces. Dans un second temps, l’identification la plus exhaustive possible de phénotypes anormaux a révélé 10 grandes catégories de malformations associées à des organes ou fonctions spécifiques. De plus, certains de ces défauts semblent fortement dépendants des conditions d’élevage des géniteurs ce qui permet d’identifier l’effet de potentiels facteurs extrinsèques sur le développement et d’améliorer les techniques de gestion des animaux. Enfin, l’ensemble de ces résultats et des paramètres mesurés durant l’embryogenèse ont permis d’effectuer une classification approfondie des pontes obtenues présentant des profils de développement similaires pour mettre en évidence des liens éventuels entre les divers phénotypes et paramètres utilisés. Les analyses de données effectuées ont montré que seulement 3 paramètres étaient nécessaires à la caractérisation de 4 profils de succès de développement variables : les taux de survie au début de l’organogenèse, d’éclosion et de malformations. A l’avenir, ces paramètres pourraient être généralisés permettant d’homogénéiser les critères d’évaluation du succès de développement chez d’autres espèces d’intérêt de poisson. L’ensemble de ces résultats constituent une base solide pour étudier l’effet des facteurs extrinsèques et/ou intrinsèques sur la qualité et le succès de développement embryonnaire / Currently, the durability of the aquaculture developmental model is clearly challenged and one solution consists to diversify the fish production by the domestication of new species such as the Eurasian perch (P. fluviatilis), a freshwater species promising and valuable for the diversification of European aquaculture. Several aspects of its reproductive biology are well known, nevertheless, only little information is available on its development. However, early developmental impairments, whose causes are unclear, actually impact the fish production quality. In this context, the present work aimed to characterize the developmental success and impairments in Eurasian perch on three main issues: (i) determine a model of normal embryogenesis table helping to (ii) define developmental impairments, in diverse rearing conditions and (iii) identify the relationships between various parameters of embryonic ontogenesis to characterize different patterns of developmental success. The first part of this study allowed identifying the accurate timing of normal ontogenesis of this species through the definition of an alternative and flexible developmental table to describe non-model fish species, allowing the intra- and inter-specific comparisons. In the second part, the exhaustive characterization of abnormal phenotypes revealed 10 categories of deformities linked to specific organs or functions. Moreover, some of these categories seemed to be related to rearing-conditions of the breeders allowing identifying the potential effects of extrinsic factors on the development and improving the management of fish. Finally, the previous results and the parameters measured during embryogenesis help to classify the several spawns obtained with the same developmental pattern and to highlight the potential relationships between diverse phenotypes and parameters. In addition, the data analyses showed that only 3 parameters are reliable to assess the developmental success: survival rate at the onset of the organogenesis, hatching and deformities rates. Henceforth, these parameters and this classification could be generalized as a new strategy to assess the developmental success in other fish species. All of these results provide a good basic knowledge to study the potential effects of various extrinsic and/or intrinsic factors on the developmental success and the embryonic quality

Historically, sperm has been considered merely as a carrier of genetic material at fertilisation. However, it is known that sperm supports embryonic development better than other cell types, suggesting that it might also have additional important, non-genetic contributions to embryonic development. The work described in this dissertation focuses on identifying the molecular determinants of developmental programming of sperm. First, the development of embryos derived from sperm and spermatids, immature precursors of sperm was compared. Sperm-derived embryos developed significantly better than spermatid-derived embryos. Further research aiming to identify the reasons for the developmental advantage of sperm led to the identification of proteins that are present specifically in sperm and not in spermatids. Moreover, egg factors which are preferentially incorporated into the sperm, but not into the spermatid chromatin were identified with the use of egg extracts, suggesting that the chromatin of sperm could be programmed to interact with the components of the egg. Subsequently, the reasons for developmental failure of spermatid-derived embryos were investigated. By comparing the sperm with spermatids it was shown that the programming of sperm to support efficient development is linked to its special ability to regulate expression of developmentally-important embryonic genes, and not to its ability to support DNA replication or rRNA production. Further characterisation of the sperm and spermatid chromatin with the use of genome-wide sequencing allowed me to link the correct regulation of gene expression in the embryo with a certain combination of epigenetic marks in the sperm, but not in the spermatid chromatin. Finally, it is shown that enzymatic removal of epigenetic modifications at fertilisation leads to misregulation of gene expression. This therefore suggests that epigenetic information contained in parental genomes at fertilisation is required for a proper regulation of embryonic transcription. My results support the hypothesis that the sperm is not only a carrier of genetic material, but also provides the embryo with epigenetic information for regulation of transcription after fertilisation. I believe that these findings advance our current understanding of the nature and mechanisms of sperm programming for embryonic development, and are important contributions to the emerging field of transgenerational inheritance of epigenetic traits in general.

Made available in DSpace on 2015-03-26T13:39:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2024686 bytes, checksum: 927a0cecb4bcec1327a0e0c20f95445e (MD5) Previous issue date: 2011-07-08 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Somatic embryogenesis is the process of development of embryos from somatic cells. The aim of this study was to evaluate the efficiency of growth regulators at different stages of somatic embryogenesis of three cultivars of sugarcane, RB 867515, RB928064 e RB925345, and characterize the effects of in vitro treatments of the anatomical development of somatic embryos. Embryogenic calluses were obtained after inoculation of immature leaves arising from lateral buds on MS medium plus 30g.L sucrose, 2.5 g.L agar and different concentrations of 2,4-D, Dicamba and CPA (5,10,15 and 20 mM). After 30 days of induction it showed that the dose of 20μM 2, 4-D was the concentration that induced the highest percentage of callus, 36.95%, and callus were compact and whitish. The calluses were subcultured in the same medium for 30 days and after this period, the callus were transferred to medium with low concentration of 2,4-D (0.5 mg.L-1) to obtain globular pro-embryos. The globular pro-embryos were transferred to multiplication medium and after 30 days were transferred to maturation medium. osmotic regulators were tested, mannitol and sucrose (30, 60 and 90 gL) and ABA (0 and 5 mM). The embryos were transferred to regeneration medium containing 1 mg. L-1 BAP and GA3. The embryos that were in the medium with 30g. L-1, with ABA when transferred to regeneration medium formed healthy and vigorous plants. The histochemical Blue Evans test and Carmine Acetic confirmed the embryogenic of the material. The anatomical study of the process of somatic embryogenesis in leaf explants of sugar cane showed the emergence of callus from the epidermis, which in more advanced stages (globular embryos) easily detached from the explant. It was evident the presence of a well-defined protoderms, isodiametric cells with prominent nuclei and nucleoli and high nucleus / cytoplasm ratio. In the later stages were observed the basal and apical meristem well defined, bounded by a protoderm and immersed in a mass of vacuolated cells, assuming one is in the process of somatic embryo formation. At the end of 30 days in maturation, it was observed a bipolar structure, consisting of stem and root apices, which had a closed vascular system, without connection to the adjacent tissue, confirming the presence of the somatic embryo. / A embriogênese somática é o processo de desenvolvimento de embriões a partir de células somáticas. O objetivo desse trabalho foi avaliar a eficiência de reguladores de crescimento nas diferentes fases da embriogênese somática de três cultivares de cana-de- açúcar, RB 867515, RB928064 e RB925345, e caracterizar os efeitos dos tratamentos in vitro no desenvolvimento anatômico dos embriões somáticos. Foram obtidos calos embriogênicos após inoculação de folhas imaturas advindas de gemas laterais em meio MS acrescido de 30g.L de sacarose, 2,5g.L de ágar e diferentes concentrações de 2,4-D, Dicamba e CPA (5,10,15 e 20 μM). Aos 30 dias após indução verificou-se que a dose de 20μM de 2,4-D foi a dose que induziu maior porcentagem de calos embriogênicos, 36,95%, sendo que estes calos tinham aspecto compacto e eram de coloração esbranquiçada. Os calos foram subcultivados no mesmo meio por mais 30 dias e após esse período, os calos embriogênicos obtidos foram transferidos para meio com baixa concentração de 2,4-D (2,26μM) para obtenção de embriões globulares. Os embriões globulares foram transferidos para meio de multiplicação composto do memso meio de indução, acrescido de três diferentes doses de AIA (6, 12 e 24 μL/placa) e foi observada diferença significativa entre as variedades testadas, sendo que na maior dose ocorreu maior multiplicação de calos embriogênicos. Após 30 dias foram transferidos para meio de maturação, onde foram testados os reguladores osmóticos, manitol e sacarose (30, 60 e 90 g.L) e o ABA (0 e 5 μM). Os embriões viáveis foram transferidos para meio de regeneração, contendo 1mg. L-1 de BAP e GA3. Os embriões somáticos que estavam em meio com 30g. L-1, com ABA quando transferidos para meio de regeneração formaram plântulas sadias e vigorosas. O teste histoquímico azul de Evans e carmim acético confirmaram a natureza embriogênica do material. O estudo anatômico do processo de embriogênese somática em explantes foliares de cana-de-açúcar evidenciou o surgimento de calos embriogênicos a partir da epiderme, que em estágios mais avançados (embriões globulares) se desprendiam facilmente do explante. Era evidente a presença de uma protoderme bem definida, células isodiamétricas, com núcleo e nucléolo proeminentes e alta relação núcleo/citoplasma. Nos estágios posteriores foi possível observar uma estrutura apresentando meristema apical e basal bem definido, delimitada por uma protoderme e imersa em uma massa de células vacuolizadas, supondo ser um embrião somático em processo de formação. Ao final dos 30 dias em meio de maturação foi observada uma estrutura bipolar, constituída de ápices caulinar e radicular, que apresentava um sistema vascular fechado, sem conexão com os tecidos adjacentes, confirmando ser um embrião somático.

Made available in DSpace on 2015-03-26T13:42:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1425522 bytes, checksum: 6c4fc6ad33dadc94d0af11b40c2fa78f (MD5) Previous issue date: 2010-09-20 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Somatic embryogenesis is a useful tool for plant propagation and a suitable model system for deciphering early events during embryo development. Molecular and genetic studies focused in plant development have reported the involvement of SERK (Somatic Embryogenesis Receptor Kinase) gene in the acquisition of embryogenic competence. In order to increase the efficiency of plant regeneration processes we need to understand it better. Therefore, the purpose of the present work was to determine if the SERK gene is present in the soybean genome and analyze its expression during somatic embryogenesis. Genes encoding tree novel members of the Leucine-rich repeat receptor like kinase (LRR-RLK) superfamily have been identified. These genes was named GmSERK1, GmSERK2 and GmSERK3, since they share a conserved intron/exon structure with other members of the SERK family and encode a signal peptide, a leucine zipper domain, five LRR, the SPP domain, a single transmembrane domain and a kinase domain with 11 subdomains, features that distinguishes SERK proteins from other proteins belonging to the LRRRLK superfamily. To investigate the position of GmSERK proteins in the plant LRR-RLKs superfamily, the conserved parts of the extracellular or intracellular domains were compared to other published LRR-RLKs sequences taken from literature. In the LRR domain, SERK proteins share all conserved amino acids of the plant LRR consensus sequence. Moreover, conservations of an aspartic acid (D) at position 1, an asparagine (N) at position 4 and a glycine (G) at position 16 were observed, although they are not consensus between other LRRs. A phylogenetic tree was constructed based on kinase domains and results showed that SERK proteins form a distinct branch in the phylogenetic tree and GmSERKs clustered most closely with SERK gene members such as Medicago truncatula (MtSERK1), ix Teobroma cacao (TcSERK) and Solanum tuberosum (StSERK1). Treatment of explants with 2,4-dichlorophenoxyacetic acid (40 mg/L) was effective in inducing somatic embryos derived from the cultivar CAC-1 and a large number of globular embryos could be viewed after 30 days. However, it was observed a strong effect of the genotype in the morphogenic response. Unlike CAC-1, explants derived from the cultivar CS303 showed a low frequency of induction and only a small number of somatic embryos were obtained. Analysis of gene expression, performed by in situ hybridization, showed that a high level of GmSERK1 transcripts could be detected in embryogenic tissues of CAC-1 during the induction of somatic embryogenesis. In contrast, immature cotyledons of soybean cultivar CS303 showed low levels of GmSERK1 transcripts. These results suggest the involvement of GmSERK1 gene with somatic embryogenesis, probably acting as an important player in acquisition of embryogenic competence. / A embriogênese somática é uma ferramenta útil para a propagação de plantas e consiste num sistema modelo adequado para a elucidação de eventos iniciais do desenvolvimento embrionário. Estudos genéticos e moleculares focados no desenvolvimento vegetal têm relatado o envolvimento do gene SERK (Somatic Embryogenesis Receptor Kinase) no processo de aquisição de competência embriogênica. Para que seja possível o aumento da eficiência dos processos de regeneração de plantas, é preciso compreendê-los melhor. Dessa forma, os objetivos do presente trabalho foram determinar se o gene SERK está presente no genoma da soja e analisar a sua expressão durante a indução de embriogênese somática. Genes que codificam três novos membros da superfamília de receptores cinase ricos em leucina (LRR-RLK - Leucine-rich repeat receptor like kinase) foram identificados. Esses genes foram denominados GmSERK1, GmSERK2 e GmSERK3, já que compartilham com outros membros da família SERK uma conservada estrutura íntron/éxon e codificam um peptídeo sinal, um zíper de leucina, cinco repetições de leucina (LRR), um domínio rico em prolina (SPP - serine proline proline), um domínio transmembrana e um domínio cinase com 11 subdomínios, características que distinguem os receptores SERK dos demais membros da superfamília LRR-RLK. Com o objetivo de verificar a posição das proteínas GmSERK dentro da superfamília de LRR-RLKs de plantas, partes conservadas de seus domínios extracelular e intracelular foram comparadas a outras sequências de LRR-RLKs retiradas da literatura. Em relação ao domínio LRR, verificou-se que as proteínas SERK apresentam todos os aminoácidos conservados da sequência consenso das LRR de plantas. Além disso, a conservação dos resíduos de ácido aspártico (D) na posição 1, asparagina (N) na posição 4 e glicina (G) na posição 16 em proteínas da família SERK foi observado, embora não seja consenso entre as LRRs de plantas. Uma árvore filogenética foi construída com base nos domínios cinase e os resultados mostraram que as proteínas SERK formam um ramo distinto dos outros tipos de receptores LRR-RLKs e as GmSERKs agrupam-se melhor com outros membros da família SERK, como as de Medicago truncatula (MtSERK1), Teobroma cacao (TcSERK) e Solanum tuberosum (StSERK1). O tratamento dos explantes com ácido 2,4-diclorofenoxiacético (40 mg/L) mostrou-se eficiente em induzir embriões somáticos a partir da cultivar CAC- 1 e um grande número de embriões globulares pode ser visualizado após 30 dias de cultivo. Entretanto, um grande efeito do genótipo na resposta morfogênica foi observado. Ao contrário de CAC-1, explantes derivados da cultivar CS303 apresentaram uma baixa freqüência de indução e apenas um pequeno número de embriões somáticos foi obtido. As análises de expressão, realizadas por meio da técnica de hibridização in situ, mostraram que um alto nível de transcritos correspondentes ao gene GmSERK1 pôde ser detectado em tecidos embriogênicos de CAC-1 durante a fase de indução de embriogênese somática. Em contraste, cotilédones imaturos da cultivar de soja CS303 apresentaram um baixo acúmulo de transcritos de GmSERK1. Estes resultados sugerem o envolvimento do gene GmSERK1 no processo de embriogênese somática, provavelmente atuando como um importante fator na aquisição de competência embriogênica.

A implantação embrionária e a placentação em cobaios são caracterizadas pela presença trofoblastos que se destacam da placenta principal, semelhantes ao trofoblasto extra-viloso de humanos. Nestes animais ultrapassam os limites, e podem ser encontrados infiltrados no profundamente no endométrio e no em ambiente externo ultrapassando aos limites da parede uterina. A cobaia desenvolve uma importante estrutura fisiológica de troca materno-embrionária, denominada de placenta vitelina invertida, definidas como membrana fetal destituída parcial ou totalmente do revestimento trofoblástico que permite a exposição do endoderma extra-embrionário em contato direto com o tecido materno. Tais características denotam um mecanismo de controle da resposta imune materna distinta dos paradigmas estabelecidos na reprodução humana e de roedores, assim como ratos e camundongos. Sendo a mais intrigante, a destituição do trofoblasto como célula da interface-materno-fetal que controla a tolerância imune-materna.No presente trabalho, procurou-se estabelecer a organização da placenta vitelina de cobaios a partir da identificação das células que compõe esta membrana extra-embrionária e identificar em que momento ocorre à remoção das células trofoblásticas, e a subseqüente forma de interação das células da placenta vitelina na interface com o tecido materno. Para tanto foram utilizados cobaias fêmeas com idade gestacional conhecida, sacrificadas para coleta de segmentos uterinos nos períodos iniciais da gestação e destinados ao processamento histotógico de embebição em parafina. Na ausência de marcadores celulares específicos conhecidos para cobaios, foram realizados testes prospectivos com reações: citoquímicas de PAS e azul de toluidina (AT; um painel de lectinas biotinadas com afinidade específica para diferentes açúcares; e imunocitoquímica para citoqueratina. As reações realizadas com PAS e AT não identificaram populações celulares com marcação seletiva. Contudo dentre as lectinas tetadas, a Erytrina cristagali lectin (ECL) apresentou reação altamente seletiva para a população de trofoblasto mural (TM) que se origina do trofectoderma, mantendo esta reatividade ao longo da gestação. Esta marcação permitiu avaliar temporal e espacialmente o destino destas células que ao longo da gestação eram mantidas como monocamada de TM revestindo externamente a placenta vitelina e, portanto, não expondo as células do endoderma parietal ou visceral ao ecido materno. Pelo acompanhamento do desenvolvimento embrionário nos cortes seriados, foi constatada no interior do blastocisto a organização de duas massas celulares internas em pólos opostos desde a fase de pré-eclosão. Uma das massas celulares constituída de embrioblastos que dará origem aos os folhetos embrionários nas fases subseqüentes, enquanto a outra formada as células tronco trofoblásticas precursora do cone ectoplacentário (CE). A cavidade da blastocele que separa estas duas massas celulares tem a sua parede revestida pelo endoderma parietal em fase tardia, após a formação da cavidade amniótica. Estes achados demonstram a pecularidade da embriogênese no cobaio, diferente daquelas descritas para humanos e outros roedores, não permitem analogias diretas, o que pode ter contribuído para o equívoco na descrição clássica da organização e constituição da placenta vitelina invertida de constituição córion-amniótica. Isto é, o trofoblasto participa da organização da placenta vitelina inicial e permanece na membrana âmnion-córion-vitelina perfazendo todo o limite do embrião ao longo da gestação. Portanto a hipótese da placenta vitelina parcial ou totalmente invertida baseada na descrição clássica em cobaios é decorrente da interpretação equivocada da embriogênese destes animais. / The guinea pig embryo implantation and placentation is characterized by trophoblast cells detaching from the main placenta in a similar way of human extra-villous trophobasts that deeply intrude inside the endometrium and sometimes also found outside the uterine wall. Furthermore, this animal also develops inverted yolk sac placenta defined as fetal membrane partially or fully devoided of trophoblast sheet that allows extra-embryonic endoderma direct exposition to the maternal environment. These characteristics denote a distinct control mechanism of maternal immune response from the established paradigm for human and rodents (rat and mouse) reproduction, being most intriguing the depriving of trophoblast as cells of maternal-fetal interface regulating the maternal immune tolerance. The present work aimed to establish the organization of guinea pig yolk sac based on identification of cell populations composing this membrane and identification if, or, when the trophoblast cells are removed from and subsequent interaction way of yolk sac cell in interface with maternal tissue. It was used pregnant guinea pig sacrificed on established gestational day to collect uterine fragments on early pregnancy stage and processed by conventional paraffin embedding. Due to absence of known specific cell markers for guinea pig, was performed the prospective evaluation using PAS and toluidine blue (TB) cytochemistry and a screening using a panel of biotinylated lectin specific for different sugars and, anti-cytokeratin. The PAS and TB staining did not identify any specific cell population, however, among the lectins used, Erytrina cristagali lectin (ECL) showed high selective labeling to the trophoblast cells originated from the trophectoderm that was kept through the gestational period. This reaction pattern was useful to evaluate chronologically and topologically the fate of this cell and confirmed the constancy of these cells layering the yolk sac placenta in contact with maternal tissue and therefore, endodermal cells were not exposed to maternal environment. Evaluation of embryo development step by step in the serial sections showed the presence of two inner cell mass in opposite sites inside the pre-hatched blastocyst. One of this, was formed with embryoblast that latter will originate the embryonic sheets and the other formed with trophoblast stem cells (ST) will originate the ectoplacental-cone. The wall of blastocele cavity separate these two inner cell mass was initially covered by a single ECL positive mural trophoblast and only later after the amniotic cavity is formed the extraembyonic endodermal cells migrate from the embryonic sheets to cover internally the blastocele cavity to organize the yolk sac placenta. These findings show the peculiarity of guinea pig embryogenesis, quite different from those described for human and rodents and therefore, does not allow direct analogy and seems to contribute in the misunderstanding of classic description of inverted yolk sac placenta and its cellular organization. It means, the trophoblast cell participates in the early organization of yolk sac placenta and remains in chorioamniotic yolk sac fetal membrane constantly limiting the embryo surface in contact with maternal environment. Therefore, the hypothesis of complete or partially inverted yolk sac placenta seems to be a miss understanding of guinea pig embryogenesis.

Orientador: Marcelo Carnier Dornelas / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T20:30:20Z (GMT). No. of bitstreams: 1 Cazoto_JulianaLacorte_M.pdf: 1128689 bytes, checksum: 95594f5893e4e8ee4fb64f1d168d493e (MD5) Previous issue date: 2009 / Resumo: Espécies do gênero Bryophyllum, pertencentes à família Crassulaceae, apresentam uma característica pouco encontrada na natureza: as regiões da margem foliar sofrem diferenciação e desenvolvem pequenas estruturas, que se desprendem da planta e dão origem a novos indivíduos. Tais formações são chamadas de 'propágulos foliares' ou 'bulbilhos'. Entretanto, o estudo da morfologia e desenvolvimento de tais estruturas permitiu que alguns autores as considerassem como embriões, e não apenas 'brotos' foliares. A formação natural de embriões somáticos é um fenômeno morfogênico restrito a poucas espécies vegetais. Em Bryophyllum, entretanto, eles parecem fazer parte da ontogenia da folha. O presente estudo caracterizou morfológica e anatomicamente essas estruturas foliares em Bryophyllum tubiflorum Harv. e Bryophyllum pinnatum Lam., nas diferentes fases de seu desenvolvimento, através de microscopia eletrônica de varredura e microscopia óptica. A expressão diferencial de homólogos aos genes WUSCHEL, MONOPTEROS, CUP SHAPED COTYLEDON, YABBY e LEAFY COTYLEDON1 também foi analisada durante o desenvolvimento dos 'propágulos', com a utilização de técnicas de hibridização in situ. Os resultados da análise do padrão de expressão gênicas, unidos aos da análise morfo-anatômica do desenvolvimento dos 'propágulos' permitem classificar essas estruturas como embriões somáticos in planta ao menos na espécie B. tubiflorum. / Abstract: Bryophyllum species from the Crassulaceae family show a rare feature: the margin of the leaf develops structures which detach from the mother plant and give rise to a new organism. These structures are known as 'leaf-plantlets' or 'bulbils'. However, morphological and developmental studies allowed some authors to classify such structures as embryos, and not just foliar buds. The natural formation of somatic embryos is a phenomenon restrict to a few plant species. Nevertheless in Bryophyllum this event seems to be part of the leaf ontogeny. This study aimed on the morphological and anatomical characterization of these leaf structures, in their different developmental stages, through scanning electronic microscopy and optical microscopy, in both Bryophyllum tubiflorum Harv. and Bryophyllum pinnatum Lam. The differential pattern of gene expression was also analyzed for the homologs of WUSCHEL, MONOPTEROS, CUP SHAPED COTYLEDON, YABBY e LEAFY COTYLEDON1 during the development of the leaf 'bulbils' by in situ hybridization. Taken together, the result of gene expression analyzes and those from morphoanatomy of plantlet development allow the classification of these structures as in planta somatic embryos, at least for B. tubiflorum. / Mestrado / Mestre em Biologia Vegetal

O experimento foi conduzido com o objetivo de avaliar a influência da interação entre o nitrato de amônio e nitrato de potássio sobre as respostas morfogênicas de embriões somáticos de pupunheiras in vitro a fim de otimizar o protocolo de micropropagação da espécie. As concentrações utilizadas foram 0, 825, 1650, 2475 e 3300 mg L-1 de nitrato e amônio e 0, 950 , 1900, 2850 e 3800 mg L-1 de nitrato de potássio, combinadas entre si, perfazendo um total de 25 tratamentos. Os tratamentos foram preparados a partir da solução completa de Murashige e Skoog, devidamente modificado para as proporções desses íons no suprimento de nitrogênio. Utilizaram-se duzentos e cinqüenta embriões somáticos com características morfológicas homogêneas, isentos de raízes e folhas, obtidos a partir de microplantas mantidas em sala de crescimento com temperatura e luminosidade controladas. Foram aferidos dados do comprimento da parte aérea e da raiz, o número de raízes, brotações e folhas, a porcentagem de ramificação da raiz, a porcentagem de raízes finas, medianas e grossas, em quatro períodos de cultura, a cada 60 dias, totalizando 240 dias de cultivo. Ao final desse período, avaliou-se também o teor de proteínas totais solúveis, o teor de clorofila pelo índice SPAD e os teores de macro e micronutrientes nas microplantas. Utilizou-se delineamento estatístico inteiramente casualizado e os dados foram submetidos ao teste de Bartlett a 5% e análise de variância (ANOVA) a 1% e 5% de probabilidade de erro ou foi elaborada uma matriz de correlação de Pearson a 1% e 5% de probabilidade de erro, conforme o caso. Os resultados permitiram inferir que aos 240 dias de cultivo as microplantas passam a investir mais em formação de parte aérea e aumentam a porcentagem de raízes finas, e funcionais. Os tratamentos que melhor favoreceram a formação de proteínas foram aqueles com concentração de 2475 mg L-1 de NH4NO3 ou com 2850 mg L-1 de KNO3. Os maiores índices SPAD ocorreram nos tratamentos com até 1650 mg L-1 de NH4NO3 combinado com até 1900 mg L-1 de KNO3. Diferentes combinações dos sais de NH4NO3 e KNO3 podem favorecer a absorção de cada nutriente. Conclui-se que os resultados obtidos no trabalho podem contribuir com a melhoria do protocolo de micropropagação de B. gasipaes, na medida em que permitem estabelecer o melhor tratamento para maximização de uma resposta específica desejada para cada momento do processo de cultivo dos embriões somáticos de pupunheiras, como enraizamento, crescimento da parte aérea, formação de proteínas e formação de brotações, facilitando dessa forma a otimização da produção de microplantas com características desejáveis e, conseqüentemente, sua aclimatização e desenvolvimento ex vitro. / This study aimed to evaluate the influence of the interaction between ammonium nitrate and potassium nitrate on the morphogenetic responses of peach palm somatic embryos in vitro cultivated, to optimize the micropropagation protocol for this species. The concentrations used were 0, 825, 1650, 2475 and 3300 mg L-1 of ammonium nitrate and 0, 950 , 1900, 2850 and 3800 mg L-1 of potassium nitrate, in all possible associations, totalizing 25 treatments. The treatments were prepared with the complete solution of Murashige and Skoog, with modified proportions of these ions in relation to the nitrogen supply. Two hundred and fifty somatic embryos with homogeneous morphological characteristics, without roots and leaves, obtained from microplants from a controlled temperature and luminosity room, were used in the experiment. Every 60 days, during 240 days, the length of shoot and root, the number of roots, propagules and leaves and the root architecture were measured. At the end of 240 days of cultivation, it was also analyzed the total soluble proteins, the foliar chlorophyll by a chlorophyll meter equipment (SPAD-502) and the macronutrients and micronutrients concentrations in the microplants. The experiment was conducted in a randomized design. The data were subjected to Bartlett´s test at 5% and variance analysis (ANOVA) at 1% and 5% of probability of error, or it was made a correlation matrix at 1% and 5% of probability of error, according to each analysis. The results showed that at 240 days of cultivation the microplants spent more energy building the shoot part and raised the percentage of thin, functional root. The best treatments for proteins formation were those with 2475 mg L-1 of NH4NO3 or with 2850 mg L-1 of KNO3. The highest SPAD index occurred in the treatments with at most 1650 mg L-1 of NH4NO3 associated with at most 1900 mg L-1 of KNO3. Different associations of NH4NO3 e KNO3 may favor the absorption of each nutrient. We conclude that the results obtained may contribute to the optimization of the B. gasipaes micropropagation protocol, as it is possible to establish the best treatment for maximization of a specific answer for each moment of the somatic embryos cultivations process, such as rooting, shoot growth, propagules and protein formation, and thus increase the optimization of microplants production with wanted characteristics and hence its acclimatization and ex vitro development.