Recent studies suggest that postmitotic neurons can reenter the cell cycle as a prelude to apoptosis after brain injury. However, most dying neurons do not pass the G 1 /S-phase checkpoint to resume DNA synthesis. The specific factors that trigger abortive DNA synthesis are not characterized. Here we show that the combination of hypoxia and ischemia induces adult rodent neurons to resume DNA synthesis as indicated by incorporation of bromodeoxyuridine (BrdU) and expression of G 1 /S-phase cell cycle transition markers. After hypoxia-ischemia, the majority of BrdU-and neuronal nuclei (NeuN)-immunoreactive cells are also terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-stained, suggesting that they undergo apoptosis. BrdU ϩ neurons, labeled shortly after hypoxia-ischemia, persist for Ͼ5 d but eventually disappear by 28 d. Before disappearing, these BrdU ϩ /NeuN ϩ / TUNEL ϩ neurons express the proliferating cell marker Ki67, lose the G 1 -phase cyclin-dependent kinase (CDK) inhibitors p16INK4 and p27Kip1 and show induction of the late G 1 /S-phase CDK2 activity and phosphorylation of the retinoblastoma protein. This contrasts to kainic acid excitotoxicity and traumatic brain injury, which produce TUNEL-positive neurons without evidence of DNA synthesis or G 1 /S-phase cell cycle transition. These findings suggest that hypoxia-ischemia triggers neurons to reenter the cell cycle and resume apoptosis-associated DNA synthesis in brain. Our data also suggest that the demonstration of neurogenesis after brain injury requires not only BrdU uptake and mature neuronal markers but also evidence showing absence of apoptotic markers. Manipulating the aberrant apoptosis-associated DNA synthesis that occurs with hypoxia-ischemia and perhaps neurodegenerative diseases could promote neuronal survival and neurogenesis.
Components of the Wnt signaling pathway are involved in patterning the sea urchin primary or animal-vegetal (AV) axis, but the molecular cues that pattern the secondary embryonic axis, the aboral/oral (AO) axis, are not known. In an analysis of signaling molecules that influence patterning along the sea urchin embryonic axes, we found that members of the activin subfamily of transforming growth factor- (
The mechanism of animal-vegetal (AV) axis formation in the sea urchin embryo is incompletely understood. Specification of the axis is thought to involve a combination of cell-cell signals and as yet unidentified maternal determinants. In Xenopus the Wnt pathway plays a crucial role in defining the embryonic axes. Recent experiments in sea urchins have shown that at least two components of the Wnt signaling pathway, GSK3beta and beta-catenin, are involved in embryonic AV axis patterning. These results support the notion that the developmental network that regulates axial patterning in deuterostomes is evolutionarily conserved. To further test this hypothesis, we have examined the role of beta-catenin nuclear binding partners, members of the TCF family of transcriptional regulators, in sea urchin AV axis patterning. To test the role of TCFs in mediating beta-catenin signals in sea urchin AV axis development we examined the consequences of microinjecting RNAs encoding altered forms of TCF on sea urchin development. We show that expression of a dominant negative TCF results in a classic "animalized" embryo. In contrast, microinjected RNA encoding an activated TCF produces a highly "vegetalized" embryo. We show that the transactivational activity of endogenous sea urchin TCF is potentiated by LiCl treatment, which vegetalizes embryos by inhibiting GSK3, consistent with an in vivo interaction between endogenous beta-catenin and TCF. We also provide evidence indicating that all of beta-catenin's activity in patterning the sea urchin AV axis is mediated by TCF. Using a glucocorticoid-responsive TCF, we show that TCF transcriptional activity affects specification along the AV axis between fertilization and the 60-cell stage.
The chick, Gallus gallus, is the traditional model in avian developmental studies. Data on other bird species are scarce. Here, we present a comparative study of the embryonic development of the chick and the emu Dromaius novaehollandiae, a member of Paleognathae, which also includes the ostrich, rhea, tinamou, kiwi, and cassowary. Emu embryos ranging from Hamburger and Hamilton (HH) equivalent stages 1 to 43 were collected and their gross morphology analyzed. Its early development was studied in detail with time-lapse imaging and molecular techniques. Emu embryos in general take 2-3 times longer incubation time to reach equivalent chicken stages, requiring 1 day for HH2, 2.5 days for HH4, 7 days for limb bud initiation, 23 days for feather germ appearance, and approximately 50-56 days for hatching. Chordin gene expression is similar in emu and chick embryos, and emu Brachyury is not expressed until HH3. Circulation is established at approximately the 27-to 30-somite stage. Forelimb buds are formed and patterned initially, but their growth is severely retarded. The size difference between an emu and a chick embryo only becomes apparent after limb bud formation. Overall, emu and chick embryogenesis proceeds through similar stages, but developmental heterochrony between these two species is widely observed.
The understanding of germ layer formation in vertebrates began with classical experimental embryology. Early in the 20th century, Spemann and Mangold (1924) identified a region of the early embryo capable of inducing an entire embryonic axis. Termed the dorsal organizer, the tissue and the activity have been shown to exist in all vertebrates examined. In mice, for example, the activity resides in a region of the gastrula embryo known as the node. Experiments by the Dutch embryologist Nieuwkoop (1967a, 1967b, 1973, 1977) showed that a signal derived from the vegetal half of the amphibian embryo is responsible for the formation of mesoderm. Nieuwkoop's results allowed the development of in vitro assays that led, in the late 1980s and early 1990s, to the identification of growth factors essential for germ layer formation. Through more recent genetic investigations in mice and zebrafish, we now know that one class of secreted growth factor, called Nodal because of its localized expression in the mouse node, is essential for formation of mesoderm and endoderm and for the morphological rearrangements that occur during gastrulation.
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