Mechanisms of Development最新文献

Establishment and movement of cytoplasmic domains is of great importance for the emergence of cell polarity, germline segregation, embryonic axis specification and correct sorting of organelles and macromolecules into different embryonic cells. The zebrafish oocyte, egg and zygote are valuable material for the study of cytoplasmic domains formation and dynamics during development. In this review we examined how cytoplasmic domains form and are relocated during zebrafish early embryogenesis. Distinct cortical cytoplasmic domains (also referred to as ectoplasm domains) form first during early oogenesis by the localization of mRNAs to the vegetal or animal poles of the oocyte or radially throughout the cortex. Cytoplasmic segregation in the late oocyte relocates non-cortical cytoplasm (endoplasm) into the preblastodisc and yolk cell. The preblastodisc is a precursor to the blastodisc, which gives rise to the blastoderm and most the future embryo. After egg activation, the blastodisc enlarges by transport of cytoplasm from the yolk cell to the animal pole, along defined pathways or streamers that include a complex cytoskeletal meshwork and cytoplasmic movement at different speeds. A powerful actin ring, assembled at the margin of the blastodisc, appears to drive the massive streaming of cytoplasm. The fact that the mechanism(s) leading to the formation and relocation of cytoplasmic domains are affected in maternal-effect mutants indicates that these processes are under maternal control. Here, we also discuss why these mutants represent outstanding genetic entry points to investigate the genetic basis of cytoplasmic segregation. Functional studies, combined with the analysis of zebrafish mutants, generated by forward and reverse genetic strategies, are expected to decipher the molecular mechanism(s) by which the maternal factors regulate cytoplasmic movements during early vertebrate development.

The developmental mechanisms that control the building of the complex head of vertebrates and particularly, facial skeletogenesis, remain poorly known. Progenitor cells derived from the embryonic neural crest (NC) are the major constituents and players of facial tissue development. Deciphering the cellular and molecular machinery that controls NC cell (NCC) differentiation into bone, cartilage, fat and other mesenchymal tissues, is thus a main issue for understanding vertebrate facial variations. In this work, we investigated the effects of fibroblast growth factor 8 (FGF8) and Sonic Hedgehog (Shh), two signaling molecules essential for craniofacial development, on the in vitro differentiation and multipotentiality of mesencephalic NCCs (MNCCs) isolated from the quail embryo. Comparison of distinct temporal treatments with FGF8 and/or Shh showed that both promoted chondrogenesis of MNCCs by increasing the amount and size of cartilage nodules. Higher rates of chondrogenesis were observed when MNCCs were treated with FGF8 during the migration phase, thus mimicking the in vivo exposure of migrating NCCs to FGF8 secreted by the isthmic brain signaling center. An in vitro cell cloning assay revealed that, after concomitant treatment with FGF8 and Shh, about 80% of NC progenitors displayed chondrogenic potential, while in untreated cultures, only 18% exhibited this potential. In addition, colony analysis showed for the first time the existence of a highly multipotent progenitor able to clonally give rise to adipocytes in addition to other cephalic NC phenotypes (i.e. glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes) (GNMFCA progenitor). This progenitor was observed only when clonal cultures were treated with both FGF8 and Shh. Several other types of multipotent cells, which generated four, five or six distinct phenotypes, accounted for 55% of the progenitors in FGF8 and Shh treated cultures, versus 13,5% in the untreated ones. Together, these data reveal an essential role for both FGF8 and Shh together in maintenance of MNCC multipotentiality by favoring the development of NC progenitors endowed with a broad array of mesectodermal potentials.

The centrosomal protein γ-tubulin is part of the cytoplasmic γ-tubulin small (γ-TuSCs) and large complexes (γ-TuRCs). Both, molecular and cellular evidence indicate that γ-tubulin plays a central role in microtubule nucleation and mitotic spindle formation. However, the molecular mechanisms of complex formation and subsequent biological roles in animal development remain unclear. Here, we used γ-tubulin gene knockdown in the zebrafish early embryo model to gain insights into its activity and cellular contribution during vertebrate embryogenesis. γ-Tubulin loss-of-function impaired γ-TuSC formation, impacting the microtubule nucleation rate in vitro. Moreover, decreased γ-tubulin synthesis caused dramatic defects in nuclear dynamics and cell cycle progression, leading to developmental arrest at the mid-gastrula stage. At the subcellular level, microtubule organization and function were altered, affecting chromosome segregation and triggering cell proliferation arrest and apoptosis. Our results suggest that de novo translated γ-tubulin participates in γ-TuSC formation required for early animal development. Importantly, formation of this complex is essential for both centrosome assembly and function, and cell proliferation. Thus, γ-TuSC integrity appears to be critical for cell cycle progression, and concomitantly, for coordinating the many distinct activities carried out by the early embryo. Our findings identify a novel role for γ-TuSC in the regulation of early vertebrate embryogenesis, providing molecular and biochemical starting points for future in depth studies of γ-tubulin functionality and its specific role in development.

The central dogma of molecular biology statically says that the information flows from DNA to messenger RNA to protein. But the recent advances in mass spectrometry and high throughput technology have helped the scientists to view RNA as little more than a courier of genetic information encoded in the DNA. The dynamics of RNA modifications in coding and non-coding RNAs are just emerging as a carrier of non-genetic information, uncovering a new layer of complexity in the regulation of gene expression and protein translation. In this review, we summarize about the current knowledge of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine (Ψ) modifications in RNA, and described how these RNA modifications are implicated in early animal development and in several human diseases.

Pluripotent stem cells (PSCs) are capable of self-renewing and producing all cell types derived from the three germ layers in response to developmental cues, constituting an important promise for regenerative medicine. Pluripotency depends on specific transcription factors (TFs) that induce genes required to preserve the undifferentiated state and repress other genes related to differentiation. The transcription machinery and regulatory components such as TFs are recruited dynamically on their target genes making it essential exploring their dynamics in living cells to understand the transcriptional output. Non-invasive and very sensitive fluorescence microscopy methods are making it possible visualizing the dynamics of TFs in living specimens, complementing the information extracted from studies in fixed specimens and bulk assays. In this work, we briefly describe the basis of these microscopy methods and review how they contributed to our knowledge of the function of TFs relevant to embryo development and cell differentiation in a variety of systems ranging from single cells to whole organisms.

Embryonic development repeatedly deploys a finite number of signaling pathways to control a multitude of processes such as patterning, growth and differentiation. Diversity in gene expression resulting from these signals depends on the epigenetic landscape as well as the network of interactions between different pathways at a given time. A third mechanism to generate diversity from a sole signal is to modify downstream pathway effectors by modulatory protein activity. The calcium-dependent calpain proteases are modulatory proteases that cleave proteins at specific sites, generating fragments, or neoproteins, with novel functions. Among calpain substrates are effectors of the Wnt and NFκB pathways, ERK pathway and ionic channel receptors, and cell cycle regulators. Loss of calpain function is associated to muscular dystrophy, deterioration of neural connections and embryonic patterning defects. Here we review the basic features of calpains, the principles that guide regulation by calpain activity, and recent literature on how calpain function controls fundamental aspects of animal development.

The neural crest (NC) is a multipotent migratory embryonic population that is formed during late gastrulation and gives rise to a wide array of derivatives, including cells from the peripheral nervous system (PNS), the craniofacial bones and cartilages, peripheral glial cells, and melanocyte cells, among others. In this work we analyzed the role of the Hedgehog signaling pathway effector gli2 in Xenopus NC. We provide evidence that the gli2 gene is expressed in the prospective, premigratory and migratory NC. The use of a specific morpholino against gli2 and the pharmacological specific inhibitor GANT61 in different experimental approaches allowed us to determine that gli2 is required for the induction and specification of NC cells as a transcriptional activator. Moreover, gli2 also acts by reducing apoptosis in the NC without affecting its cell proliferation status. We also demonstrated that gli2 is required cell-autonomously for NC migration, and for the formation of NC derivatives such as the craniofacial cartilages, melanocytes and the cranial ganglia. Altogether, our results showed that gli2 is a key transcriptional activator to accomplish the proper specification and development of Xenopus NC cells.

Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5′-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea, and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos.

The frog neuromuscular junction (NMJ) has been extensively used as a model system to dissect the mechanisms involved in synapse formation, maturation, maintenance, regeneration, and function. Early NMJ synaptogenesis relies on a combination of cell-autonomous and interdependent pre/postsynaptic communication processes. Due to their transparency, comparatively easy manipulation, and remarkable regenerative abilities, frog tadpoles constitute an excellent model to study NMJ formation and regeneration. Here, we aimed to contribute new aspects on the characterization of the ontogeny of NMJ formation in Xenopus embryos and to explore the morphological changes occurring at the NMJ after spinal cord injury. Following analyses of X. tropicalis tadpoles during development we found that the early pathfinding of rostral motor axons is likely helped by previously formed postsynaptic specializations, whereas NMJ formation in recently differentiated ventral muscles in caudal segments seems to rely on presynaptic inputs. After spinal cord injury of X. laevis tadpoles our results suggest that rostral motor axon projections help caudal NMJ re-innervation before spinal cord connectivity is repaired.