RESUMO
Uterine implantation is a critical element of mammalian reproduction and is a tightly and highly coordinated event. An intricate and reciprocal uterine-embryo dialog exists to synchronize uterine receptivity with the concomitant activation of the blastocyst, maximizing implantation success. While a number of pathways involved in regulating uterine receptivity have been identified in the mouse, less is understood about blastocyst activation, the process by which the trophectoderm (TE) receives extrinsic cues that initiate new characteristics essential for implantation. Amino acids (AA) have been found to regulate blastocyst activation and TE motility in vitro. In particular, we find that arginine and leucine alone are necessary and sufficient to induce TE motility. Both arginine and leucine act individually and additively to propagate signals that are dependent on the activity of the mammalian target of rapamycin complex 1 (mTORC1). The activities of the well-established downstream targets of mTORC1, p70S6K and 4EBP, do not correlate with trophoblast motility, suggesting that an independent-rapamycin-sensitive pathway operates to induce trophoblast motility, or that other, parallel amino acid-dependent pathways are also involved. We find that endogenous uterine factors act to induce mTORC1 activation and trophoblast motility at a specific time during pregnancy, and that this uterine signal is later than the previously defined signal that induces the attachment reaction. In vivo matured blastocysts exhibit competence to respond to an 8-hour AA stimulus by activating mTOR and subsequently undergoing trophoblast outgrowth by the morning of day 4.5 of pregnancy, but not on day 3.5. By the late afternoon of day 4.5, the embryos no longer require any exposure to AA to undergo trophoblast outgrowth in vitro, demonstrating the existence and timing of an equivalent in vivo signal. These results suggest that there are two separate uterine signals regulating implantation, one that primes the embryo for the attachment reaction and another that activates mTOR and initiates invasive behavior.
Assuntos
Arginina/farmacologia , Blastocisto/citologia , Blastocisto/enzimologia , Movimento Celular/efeitos dos fármacos , Leucina/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Trofoblastos/citologia , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Blastocisto/efeitos dos fármacos , Gonadotropina Coriônica/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Complexos Multiproteicos , Fosforilação/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Trofoblastos/efeitos dos fármacosRESUMO
BACKGROUND: During gastrulation, an embryo acquires the three primordial germ layers that will give rise to all of the tissues in the body. In amniote embryos, this process occurs via an epithelial to mesenchymal transition (EMT) of epiblast cells at the primitive streak. Although the primitive streak is vital to development, many aspects of how it forms and functions remain poorly understood. RESULTS: Using live, 4 dimensional imaging and immunohistochemistry, we have shown that the posterior epiblast of the pre-streak murine embryo does not display convergence and extension behavior or large scale migration or rearrangement of a cell population. Instead, the primitive streak develops in situ and elongates by progressive initiation EMT in the posterior epiblast. Loss of basal lamina (BL) is the first step of this EMT, and is strictly correlated with ingression of nascent mesoderm. Once the BL is lost in a given region, cells leave the epiblast by apical constriction in order to enter the primitive streak. CONCLUSIONS: This is the first description of dynamic cell behavior during primitive streak formation in the mouse embryo, and reveals mechanisms that are quite distinct from those observed in other amniote model systems. Unlike chick and rabbit, the murine primitive streak arises in situ by progressive initiation of EMT beginning in the posterior epiblast, without large-scale movement or convergence and extension of epiblast cells.
Assuntos
Transição Epitelial-Mesenquimal , Linha Primitiva/citologia , Linha Primitiva/fisiologia , Animais , Movimento Celular , Camadas Germinativas/citologia , Camadas Germinativas/fisiologia , Camundongos , Camundongos Endogâmicos , Transdução de SinaisRESUMO
We review what is known about amphibian limb regeneration from the prospective of developing strategies for the induction of regeneration in adult mammals. Prominent in urodele amphibian limb regeneration is the formation of a blastema of undifferentiated cells that goes on to reform the limb. The blastema shares many properties with the developing limb bud; thus, the outgrowth phase of regeneration can be thought of as cells going through development again, i.e., redevelopment. Getting to a redevelopment phase in mammals would be a major breakthrough given our extensive understanding of limb development. The formation of the blastema itself represents a transition phase in which limb cells respond to injury by dedifferentiating to become embryonic limb progenitor cells that can undergo redevelopment. During this phase, rapid wound closure is followed by the dedifferentiation of limb cells to form the blastema. Thus, the regeneration process can be divided into a wound-healing/dedifferentiation phase and a redevelopment phase, and we propose that the interface between the wound-healing response and gaining access to developmentally regulated programs (dedifferentiation) lies at the heart of the regeneration problem in mammals. In urodele amphibians, dedifferentiation can occur in all of the tissues of the limb; however, numerous studies lead us to focus on the epidermis, the dermis, and muscle as key regulators of regeneration. Among higher vertebrates, the digit tip in mammals, including humans, is regeneration-competent and offers a unique mammalian model for regeneration. Recent genetic studies in mice identify the Msx1 gene as playing a critical role in the injury response leading to digit tip regeneration. The results from regeneration studies ranging from amphibians to mammals can be integrated to develop a roadmap for mammalian regeneration that has as its focus understanding the phenomenon of dedifferentiation.
Assuntos
Extremidades/fisiologia , Regeneração/fisiologia , Vertebrados/fisiologia , Animais , Cartilagem/fisiologia , Derme/fisiologia , Extremidades/lesões , Humanos , Músculos/fisiologiaRESUMO
Despite being implicated as a mechanism driving gastrulation and body axis elongation in mouse embryos, the cellular mechanisms underlying mammalian convergent extension (CE) are unknown. Here we show, with high-resolution time-lapse imaging of living mouse embryos, that mesodermal CE occurs by mediolateral cell intercalation, driven by mediolaterally polarized cell behavior. The initial events in the onset of CE are mediolateral elongation, alignment and orientation of mesoderm cells as they exit the primitive streak. This cell shape change occurs prior to, and is required for, the subsequent onset of mediolaterally polarized protrusive activity. In embryos mutant for PTK7, a novel cell polarity protein, the normal cell elongation and alignment upon leaving the primitive streak, the subsequent polarized protrusive activity, and CE and axial elongation all failed. The mesoderm normally thickens and extends, but on failure of convergence movements in Ptk7 mutants, the mesoderm underwent radial intercalation and excessive thinning, which suggests that a cryptic radial cell intercalation behavior resists excessive convergence-driven mesodermal thickening in normal embryos. When unimpeded by convergence forces in Ptk7 mutants, this unopposed radial intercalation resulted in excessive thinning of the mesoderm. These results show for the first time the polarized cell behaviors underlying CE in the mouse, demonstrate unique aspects of these behaviors compared with those of other vertebrates, and clearly define specific roles for planar polarity and for the novel planar cell polarity gene, Ptk7, as essential regulators of mediolateral cell intercalation during mammalian CE.
Assuntos
Padronização Corporal/fisiologia , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Gastrulação/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Embrião de Mamíferos/fisiologia , Mesoderma/fisiologia , Camundongos , Mutação , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Pigpen, a nuclear protein with RNA-binding motifs and a putative transcriptional activation domain (TAD), is expressed at high levels in proliferating endothelial cells and expression is down-regulated when cells adopt a quiescent or differentiated phenotype. We cloned the mouse homolog of pigpen and investigated the regulation of its expression during embryogenesis. In situ hybridization demonstrated that a broad pattern of pigpen expression became restricted during tooth formation in the mandible. In the eye, pigpen showed a spatial restriction to the more proliferating and less differentiated regions of the lens and neural retina. Expression was also restricted in the developing vibrissae, lung, and kidney, all sites where epithelial-mesenchymal interactions are vital for morphogenesis. In vitro assays, that focused on the mandible and tooth development, indicated that epithelial signals, mediated by fibroblast growth factor-8, were required to maintain pigpen expression in the mandibular mesenchyme, whereas bone morphogenetic protein-4 negatively regulated expression in that tissue during early odontogenesis. At the protein level, immunocytochemistry demonstrated that Pigpen was expressed diffusely in the cytoplasm and more concentratedly in focal granules within the nuclei of mouse embryonic cells. Lastly, CAT reporter assays showed that the N-terminus of mouse pigpen encodes an active TAD. These data suggest that mouse Pigpen may activate transcription in vivo in response to specific growth factor signals and regulate proliferation and/or differentiation events during mouse organogenesis.