O objetivo deste estudo foi caracterizar morfologicamente o desenvolvimento inicial da gestação bovina proveniente de monta natural, com idades compreendidas entre 15 a 70 dias, e de fecundação in vitro (FIV) com 35 dias, com ênfase no desenvolvimento placentário inicial, e diferenciação das estruturas extra embrionárias. Para tanto, foram utilizados 141 conceptos, provenientes de monta natural, e sete conceptos obtidos pela técnica de FIV. Após as coletas, os conceptos foram dissecados, mensurados macroscopicamente e fotodocumentados. As membranas extra-embrionárias foram cortadas em fragmentos de 5 cm2, e, em seguida fixadas em paraformoldeído 4%, para análise por microscopia de luz, e glutaraldeído 2,5%, para utilização em microscopia eletrônica de varredura e transmissão. As membranas extra-embrionárias e fetais apresentaram graus variáveis de desenvolvimento ao longo dos períodos analisados. O aparecimento macroscópico da vascularização do alantóide, sua tentativa de se fundir com o cório e o aparecimento efetivo dos primeiros cotilédones em desenvolvimento, foram eventos observados em embriões a partir de 1,9±0,27 cm de \"Crown-Rump\" (CR) (30 a 40 dias da gestação). O CR médio, o peso do embrião, o peso do saco gestacional e os comprimentos do cório e âmnio aumentaram gradativamente com o evoluir da gestação. O epitélio alantoidiano apresentou um dimorfismo celular a partir de 0,9 cm de CR (15 a 20 dias de gestação), porém, mostrou-se imaturo até o feto atingir o comprimento de 7,2 cm de CR (60 a 70 dias da gestação). O trofoblasto apresentou células mononucleadas e células gigantes binucleadas em diferentes níveis ao longo da gestação. O saco vitelino persistiu até 70 dias de gestação, e o seu epitélio apresentou indícios de atividade funcional até 50 dias de gestação. De todos os parâmetros mensurados na análise macroscópica, somente o comprimento o CR e o saco vitelino apresentaram diferença significativa entre os conceptos de monta natural e de FIV. Nos conceptos de monta natural, o comprimento do saco vitelino foi de 5,53 cm, em média, e nos conceptos de FIV, de apenas 1,07 cm. Todavia, faz-se necessário analisar um número maior de animais submetidos a FIV para corroborar a diferença encontrada nestas medidas. Os resultados sugerem ainda a existência de uma placenta vitelínica ativa, importante para a manutenção da gestação, que se estabelece temporariamente entre a placenta coriovitelínica e alantovitelínica transitória e a placenta cório-alantóide definitiva. / The main goal of this study was to describe morphology and the early gestational development of 15 to 70 day-old bovine embryos obtained by natural mating and 35 day-old bovine embryos obtained by in vitro fertilization (IFV) technique. One hundred and forty-one concepts originated by natural mating and seven by IVF technique were used. All concepts were dissected, macroscopically measured and photographed. Extraembryonic membranes were cut in 5 cm2 fragments and fixed in 4% paraformoldehyde for light and Scanning Electron Microscopy (SEM) and in 2.5% glutaraldehyde for Transmition Electron Microscopy (TEM). Ali membranes showed different stages of development during analyzed periods. The beginning of allantois macroscopic vascularization, the attempt of fusion between allantois and chorium membranes and the effective development of the first cotyledons were observed in 30-to-40 day-old embryos with 1.9± 0.24 cm of Crown-Rump (CR) length. The average CR, the embryos and gestational sac weight, the chorion and amnion length increased during gestation. The allantoic epithelia showed a cellular dimorphism with 0.9 em CR, however, the maturation has not happened until the foetal length of 7.2 em CR. The trophoblast showed different levels of mononucleate cells and binucleate giant cells. The yolk sac persisted until 70 days of gestation and the epithelium seemed functionally activated until 50 days of gestation. The CR and yolk sac size were the only measured macroscopic parameter showingdifference between natural mating and FIV concepts. Yolk sac size presented average values of 5.53 cm and 1.07 cm for natural mating and FIV concepts, respectively. Therefore, it is necessary to analyze a higher number of FIV embryos to corroborate that observed yolk sac size difference. All results obtained in this study suggest the existent of an active vitelline placenta, important to the gestational maintenance and temporarily establish between the transitory choriovitelline placenta and the permanent chorioallantoic placenta.

O objetivo deste estudo foi caracterizar morfologicamente o desenvolvimento inicial da gestação bovina proveniente de monta natural, com idades compreendidas entre 15 a 70 dias, e de fecundação in vitro (FIV) com 35 dias, com ênfase no desenvolvimento placentário inicial, e diferenciação das estruturas extra embrionárias. Para tanto, foram utilizados 141 conceptos, provenientes de monta natural, e sete conceptos obtidos pela técnica de FIV. Após as coletas, os conceptos foram dissecados, mensurados macroscopicamente e fotodocumentados. As membranas extra-embrionárias foram cortadas em fragmentos de 5 cm2, e, em seguida fixadas em paraformoldeído 4%, para análise por microscopia de luz, e glutaraldeído 2,5%, para utilização em microscopia eletrônica de varredura e transmissão. As membranas extra-embrionárias e fetais apresentaram graus variáveis de desenvolvimento ao longo dos períodos analisados. O aparecimento macroscópico da vascularização do alantóide, sua tentativa de se fundir com o cório e o aparecimento efetivo dos primeiros cotilédones em desenvolvimento, foram eventos observados em embriões a partir de 1,9±0,27 cm de \"Crown-Rump\" (CR) (30 a 40 dias da gestação). O CR médio, o peso do embrião, o peso do saco gestacional e os comprimentos do cório e âmnio aumentaram gradativamente com o evoluir da gestação. O epitélio alantoidiano apresentou um dimorfismo celular a partir de 0,9 cm de CR (15 a 20 dias de gestação), porém, mostrou-se imaturo até o feto atingir o comprimento de 7,2 cm de CR (60 a 70 dias da gestação). O trofoblasto apresentou células mononucleadas e células gigantes binucleadas em diferentes níveis ao longo da gestação. O saco vitelino persistiu até 70 dias de gestação, e o seu epitélio apresentou indícios de atividade funcional até 50 dias de gestação. De todos os parâmetros mensurados na análise macroscópica, somente o comprimento o CR e o saco vitelino apresentaram diferença significativa entre os conceptos de monta natural e de FIV. Nos conceptos de monta natural, o comprimento do saco vitelino foi de 5,53 cm, em média, e nos conceptos de FIV, de apenas 1,07 cm. Todavia, faz-se necessário analisar um número maior de animais submetidos a FIV para corroborar a diferença encontrada nestas medidas. Os resultados sugerem ainda a existência de uma placenta vitelínica ativa, importante para a manutenção da gestação, que se estabelece temporariamente entre a placenta coriovitelínica e alantovitelínica transitória e a placenta cório-alantóide definitiva. / The main goal of this study was to describe morphology and the early gestational development of 15 to 70 day-old bovine embryos obtained by natural mating and 35 day-old bovine embryos obtained by in vitro fertilization (IFV) technique. One hundred and forty-one concepts originated by natural mating and seven by IVF technique were used. All concepts were dissected, macroscopically measured and photographed. Extraembryonic membranes were cut in 5 cm2 fragments and fixed in 4% paraformoldehyde for light and Scanning Electron Microscopy (SEM) and in 2.5% glutaraldehyde for Transmition Electron Microscopy (TEM). Ali membranes showed different stages of development during analyzed periods. The beginning of allantois macroscopic vascularization, the attempt of fusion between allantois and chorium membranes and the effective development of the first cotyledons were observed in 30-to-40 day-old embryos with 1.9± 0.24 cm of Crown-Rump (CR) length. The average CR, the embryos and gestational sac weight, the chorion and amnion length increased during gestation. The allantoic epithelia showed a cellular dimorphism with 0.9 em CR, however, the maturation has not happened until the foetal length of 7.2 em CR. The trophoblast showed different levels of mononucleate cells and binucleate giant cells. The yolk sac persisted until 70 days of gestation and the epithelium seemed functionally activated until 50 days of gestation. The CR and yolk sac size were the only measured macroscopic parameter showingdifference between natural mating and FIV concepts. Yolk sac size presented average values of 5.53 cm and 1.07 cm for natural mating and FIV concepts, respectively. Therefore, it is necessary to analyze a higher number of FIV embryos to corroborate that observed yolk sac size difference. All results obtained in this study suggest the existent of an active vitelline placenta, important to the gestational maintenance and temporarily establish between the transitory choriovitelline placenta and the permanent chorioallantoic placenta.

We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell, and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition, and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order, and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid-filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates—the amniote egg presenting one “solution” that enabled the conquest of land for reproduction.

La famille des gènes Hox code pour des facteurs de transcription connus pour leur contribution essentielle à l’élaboration de l’architecture du corps et ce, au sein de tout le règne animal. Au cours de l’évolution chez les vertébrés, les gènes Hox ont été redéfinis pour générer toute une variété de nouveaux tissus/organes. Souvent, cette diversification s’est effectuée via des changements quant au contrôle transcriptionnel des gènes Hox. Chez les mammifères, la fonction de Hoxa13 n’est pas restreinte qu’à l’embryon même, mais s’avère également essentielle pour le développement de la vascularisation fœtale au sein du labyrinthe placentaire, suggérant ainsi que sa fonction au sein de cette structure aurait accompagné l’émergence des espèces placentaires. Au chapitre 2, nous mettons en lumière le recrutement de deux autres gènes Hoxa, soient Hoxa10 et Hoxa11, au compartiment extra-embryonnaire. Nous démontrons que l’expression de Hoxa10, Hoxa11 et Hoxa13 est requise au sein de l’allantoïde, précurseur du cordon ombilical et du système vasculaire fœtal au sein du labyrinthe placentaire. De façon intéressante, nous avons découvert que l’expression des gènes Hoxa10-13 dans l’allantoïde n’est pas restreinte qu’aux mammifères placentaires, mais est également présente chez un vertébré non-placentaire, indiquant que le recrutement des ces gènes dans l’allantoïde précède fort probablement l’émergence des espèces placentaires. Nous avons généré des réarrangements génétiques et utilisé des essais transgéniques pour étudier les mécanismes régulant l’expression des gènes Hoxa dans l’allantoïde. Nous avons identifié un fragment intergénique de 50 kb capable d’induire l’expression d’un gène rapporteur dans l’allantoïde. Cependant, nous avons trouvé que le mécanisme de régulation contrôlant l’expression du gène Hoxa au sein du compartiment extra-embryonnaire est fort complexe et repose sur plus qu’un seul élément cis-régulateur. Au chapitre 3, nous avons utilisé la cartographie génétique du destin cellulaire pour évaluer la contribution globale des cellules exprimant Hoxa13 aux différentes structures embryonnaires. Plus particulièrement, nous avons examiné plus en détail l’analyse de la cartographie du destin cellulaire de Hoxa13 dans les pattes antérieures en développement. Nous avons pu déterminer que, dans le squelette du membre, tous les éléments squelettiques de l’autopode (main), à l’exception de quelques cellules dans les éléments carpiens les plus proximaux, proviennent des cellules exprimant Hoxa13. En contraste, nous avons découvert que, au sein du compartiment musculaire, les cellules exprimant Hoxa13 et leurs descendantes (Hoxa13lin+) s’étendent à des domaines plus proximaux du membre, où ils contribuent à générer la plupart des masses musculaires de l’avant-bras et, en partie, du triceps. De façon intéressante, nous avons découvert que les cellules exprimant Hoxa13 et leurs descendantes ne sont pas distribuées uniformément parmi les différents muscles. Au sein d’une même masse musculaire, les fibres avec une contribution Hoxa13lin+ différente peuvent être identifiées et les fibres avec une contribution semblable sont souvent regroupées ensemble. Ce résultat évoque la possibilité que Hoxa13 soit impliqué dans la mise en place de caractéristiques spécifiques des groupes musculaires, ou la mise en place de connections nerf-muscle. Prises dans leur ensemble, les données ici présentées permettent de mieux comprendre le rôle de Hoxa13 au sein des compartiments embryonnaires et extra-embryonnaires. Par ailleurs, nos résultats seront d’une importance primordiale pour soutenir les futures études visant à expliquer les mécanismes transcriptionnels soutenant la régulation des gènes Hoxa dans les tissus extra-embryonnaires. / The Hox family of transcription factors is well known for its key contribution in the establishment of the body architecture in all the animal kingdom. During vertebrate evolution, Hox genes have been co-opted to pattern a variety of novel tissues/organs. Often, this diversification has been achieved by changes in Hox transcriptional control. In mammals, Hoxa13 function is not restricted to the embryo proper, but is also essential for the proper development of the fetal vasculature within the placental labyrinth, suggesting that its function in this structure accompanied the emergence of placental species. In chapter 2, we report on the recruitment of two other Hoxa genes, namely Hoxa10 and Hoxa11, in the extra embryonic compartment. We show that Hoxa10, Hoxa11 and Hoxa13 expression is required in the allantois, the precursor of the umbilical cord and fetal vasculature within the placental labyrinth. Interestingly, we found that Hoxa10-13 gene expression in the allantois is not restricted to placental mammals, but is also present in a non-placental vertebrate, indicating that the recruitment of these genes in the allantois most likely predates the emergence of placental species. We generated genetic rearrangements and used transgenic assays to investigate the regulatory mechanisms underlying Hoxa gene expression in the allantois. We identified a 50 kb intergenic fragment able to drive reporter gene expression in the allantois. However, we found that the regulatory mechanism controlling Hoxa gene expression in the extra-embryonic compartment is very complex and relies on more than one cis-regulatory element. In chapter 3, we used genetic fate mapping to assess the overall contribution of Hoxa13 expressing cells to the different embryonic structures. In particular, we focused on Hoxa13 fate-mapping analysis in the developing forelimbs. We could determine that, in the limb skeleton, all autopod (hand) skeletal elements, with the exception of a few cells in the most proximal carpal elements, originate from Hoxa13 expressing cells. In contrast, we found that, in the muscle compartment, Hoxa13 expressing cells and their descendants extend to more proximal limb domains, where they contribute to most of the muscle masses of the forearm and, in part, to the triceps. Interestingly we found that Hoxa13 expressing cells and their descendants are not identically distributed among different muscles. Within the same muscular mass, fibres with different Hoxa13lin+ contribution can be identified, and fibers with similar contribution are often clustered together. This result raises the possibility that Hoxa13 might be involved in establishing specific features of muscle groups, or in establishing nerve-muscle connectivity. Altogether, the data presented herein provide a better understanding of the role of Hoxa13 in both the embryonic and extra-embryonic compartment. Moreover, our results will be of key importance for further investigations aimed at unravelling transcriptional mechanisms underlying Hoxa gene regulation in extra embryonic tissues.

La famille des gènes Hox code pour des facteurs de transcription connus pour leur contribution essentielle à l’élaboration de l’architecture du corps et ce, au sein de tout le règne animal. Au cours de l’évolution chez les vertébrés, les gènes Hox ont été redéfinis pour générer toute une variété de nouveaux tissus/organes. Souvent, cette diversification s’est effectuée via des changements quant au contrôle transcriptionnel des gènes Hox. Chez les mammifères, la fonction de Hoxa13 n’est pas restreinte qu’à l’embryon même, mais s’avère également essentielle pour le développement de la vascularisation fœtale au sein du labyrinthe placentaire, suggérant ainsi que sa fonction au sein de cette structure aurait accompagné l’émergence des espèces placentaires. Au chapitre 2, nous mettons en lumière le recrutement de deux autres gènes Hoxa, soient Hoxa10 et Hoxa11, au compartiment extra-embryonnaire. Nous démontrons que l’expression de Hoxa10, Hoxa11 et Hoxa13 est requise au sein de l’allantoïde, précurseur du cordon ombilical et du système vasculaire fœtal au sein du labyrinthe placentaire. De façon intéressante, nous avons découvert que l’expression des gènes Hoxa10-13 dans l’allantoïde n’est pas restreinte qu’aux mammifères placentaires, mais est également présente chez un vertébré non-placentaire, indiquant que le recrutement des ces gènes dans l’allantoïde précède fort probablement l’émergence des espèces placentaires. Nous avons généré des réarrangements génétiques et utilisé des essais transgéniques pour étudier les mécanismes régulant l’expression des gènes Hoxa dans l’allantoïde. Nous avons identifié un fragment intergénique de 50 kb capable d’induire l’expression d’un gène rapporteur dans l’allantoïde. Cependant, nous avons trouvé que le mécanisme de régulation contrôlant l’expression du gène Hoxa au sein du compartiment extra-embryonnaire est fort complexe et repose sur plus qu’un seul élément cis-régulateur. Au chapitre 3, nous avons utilisé la cartographie génétique du destin cellulaire pour évaluer la contribution globale des cellules exprimant Hoxa13 aux différentes structures embryonnaires. Plus particulièrement, nous avons examiné plus en détail l’analyse de la cartographie du destin cellulaire de Hoxa13 dans les pattes antérieures en développement. Nous avons pu déterminer que, dans le squelette du membre, tous les éléments squelettiques de l’autopode (main), à l’exception de quelques cellules dans les éléments carpiens les plus proximaux, proviennent des cellules exprimant Hoxa13. En contraste, nous avons découvert que, au sein du compartiment musculaire, les cellules exprimant Hoxa13 et leurs descendantes (Hoxa13lin+) s’étendent à des domaines plus proximaux du membre, où ils contribuent à générer la plupart des masses musculaires de l’avant-bras et, en partie, du triceps. De façon intéressante, nous avons découvert que les cellules exprimant Hoxa13 et leurs descendantes ne sont pas distribuées uniformément parmi les différents muscles. Au sein d’une même masse musculaire, les fibres avec une contribution Hoxa13lin+ différente peuvent être identifiées et les fibres avec une contribution semblable sont souvent regroupées ensemble. Ce résultat évoque la possibilité que Hoxa13 soit impliqué dans la mise en place de caractéristiques spécifiques des groupes musculaires, ou la mise en place de connections nerf-muscle. Prises dans leur ensemble, les données ici présentées permettent de mieux comprendre le rôle de Hoxa13 au sein des compartiments embryonnaires et extra-embryonnaires. Par ailleurs, nos résultats seront d’une importance primordiale pour soutenir les futures études visant à expliquer les mécanismes transcriptionnels soutenant la régulation des gènes Hoxa dans les tissus extra-embryonnaires. / The Hox family of transcription factors is well known for its key contribution in the establishment of the body architecture in all the animal kingdom. During vertebrate evolution, Hox genes have been co-opted to pattern a variety of novel tissues/organs. Often, this diversification has been achieved by changes in Hox transcriptional control. In mammals, Hoxa13 function is not restricted to the embryo proper, but is also essential for the proper development of the fetal vasculature within the placental labyrinth, suggesting that its function in this structure accompanied the emergence of placental species. In chapter 2, we report on the recruitment of two other Hoxa genes, namely Hoxa10 and Hoxa11, in the extra embryonic compartment. We show that Hoxa10, Hoxa11 and Hoxa13 expression is required in the allantois, the precursor of the umbilical cord and fetal vasculature within the placental labyrinth. Interestingly, we found that Hoxa10-13 gene expression in the allantois is not restricted to placental mammals, but is also present in a non-placental vertebrate, indicating that the recruitment of these genes in the allantois most likely predates the emergence of placental species. We generated genetic rearrangements and used transgenic assays to investigate the regulatory mechanisms underlying Hoxa gene expression in the allantois. We identified a 50 kb intergenic fragment able to drive reporter gene expression in the allantois. However, we found that the regulatory mechanism controlling Hoxa gene expression in the extra-embryonic compartment is very complex and relies on more than one cis-regulatory element. In chapter 3, we used genetic fate mapping to assess the overall contribution of Hoxa13 expressing cells to the different embryonic structures. In particular, we focused on Hoxa13 fate-mapping analysis in the developing forelimbs. We could determine that, in the limb skeleton, all autopod (hand) skeletal elements, with the exception of a few cells in the most proximal carpal elements, originate from Hoxa13 expressing cells. In contrast, we found that, in the muscle compartment, Hoxa13 expressing cells and their descendants extend to more proximal limb domains, where they contribute to most of the muscle masses of the forearm and, in part, to the triceps. Interestingly we found that Hoxa13 expressing cells and their descendants are not identically distributed among different muscles. Within the same muscular mass, fibres with different Hoxa13lin+ contribution can be identified, and fibers with similar contribution are often clustered together. This result raises the possibility that Hoxa13 might be involved in establishing specific features of muscle groups, or in establishing nerve-muscle connectivity. Altogether, the data presented herein provide a better understanding of the role of Hoxa13 in both the embryonic and extra-embryonic compartment. Moreover, our results will be of key importance for further investigations aimed at unravelling transcriptional mechanisms underlying Hoxa gene regulation in extra embryonic tissues.

No description available.