n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and 5-ethynyl-2--deoxyuridine

MicroRNAs (miRNAs) are 19-25 nucleotide RNAs that regulate messenger RNA translation and stability. Recently, we performed a conditional knockout (CKO) of the miRNA-processing enzyme Dicer during mouse retinal development and showed an essential role for miRNAs in the transition of retinal progenitors from an early to a late competence state (Georgi and Reh [2010]: J Neurosci 30:4048-4061). Notably, Dicer CKO progenitors failed to express Ascl1 and generated ganglion cells beyond their normal competence window. Because Ascl1 regulates multiple Notch signaling components, we hypothesized that Notch signaling is downregulated in Dicer CKO retinas. We show here that Notch signaling is severely reduced in Dicer CKO retinas, but that retinal progenitors still retain a low level of Notch signaling. By increasing Notch signaling in Dicer CKO progenitors through constitutive expression of the Notch intracellular domain (NICD), we show that transgenic rescue of Notch signaling has little effect on the competence of retinal progenitors or the enhanced generation of ganglion cells, suggesting that loss of Notch signaling is not a major determinant of these phenotypes. Nevertheless, transgenic NICD expression restored horizontal cells, suggesting an interaction between miRNAs and Notch signaling in the development of this cell type. Furthermore, while NICD overexpression leads to robust glial induction in control retinas, NICD overexpression was insufficient to drive Dicer-null retinal progenitors to a glial fate. Surprisingly, the presence of transgenic NICD expression did not prevent the differentiation of some types of retinal neurons, suggesting that Notch inactivation is not an absolute requirement for the initial stages of neuronal differentiation.

Topics: Animals; Animals, Newborn; Antimetabolites; Bacterial Proteins; Cell Differentiation; DEAD-box RNA Helicases; Deoxyuridine; Dipeptides; Enzyme Inhibitors; Eye Proteins; Gene Expression Regulation, Developmental; Homeodomain Proteins; Luminescent Proteins; Mice; Organ Culture Techniques; Paired Box Transcription Factors; PAX6 Transcription Factor; Receptors, Notch; Repressor Proteins; Retina; Ribonuclease III; Signal Transduction; Sphincterotomy, Transduodenal; Stem Cells

Dopaminergic neurons develop at distinct anatomical sites to form some of the major neuromodulatory systems in the vertebrate brain. Despite their relevance in neurodegenerative diseases and the interests in reconstitutive therapies from stem cells, mechanisms of the neurogenic switch from precursor populations to dopaminergic neurons are not well understood. Here, we investigated neurogenesis of different dopaminergic and noradrenergic neuron populations in the zebrafish embryo. Birth-dating analysis by EdU (5-ethynyl-2'-deoxyuridine) incorporation revealed temporal dynamics of catecholaminergic neurogenesis. Analysis of Notch signaling mutants and stage-specific pharmacological inhibition of Notch processing revealed that dopaminergic neurons form by temporally distinct mechanisms: dopaminergic neurons of the posterior tuberculum derive directly from neural plate cells during primary neurogenesis, whereas other dopaminergic groups form in continuous or wavelike neurogenesis phases from proliferating precursor pools. Systematic analysis of Notch ligands revealed that the two zebrafish co-orthologs of mammalian Delta1, DeltaA and DeltaD, control the neurogenic switch of all early developing dopaminergic neurons in a partially redundant manner. DeltaA/D may also be involved in maintenance of dopaminergic precursor pools, as olig2 expression in ventral diencephalic dopaminergic precursors is affected in dla/dld mutants. DeltaA/D act upstream of sim1a and otpa during dopaminergic specification. However, despite the fact that both dopaminergic and corticotropin-releasing hormone neurons derive from sim1a- and otpa-expressing precursors, DeltaA/D does not act as a lineage switch between these two neuronal types. Rather, DeltaA/D limits the size of the sim1a- and otpa-expressing precursor pool from which dopaminergic neurons differentiate.

Topics: Animals; Animals, Genetically Modified; Basic Helix-Loop-Helix Transcription Factors; Brain; Cell Cycle; Corticotropin-Releasing Hormone; Deoxyuridine; Dipeptides; Dopamine; Embryo, Nonmammalian; Enzyme Inhibitors; Gene Expression Regulation, Developmental; HSP70 Heat-Shock Proteins; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; Nerve Tissue Proteins; Neurogenesis; Oligodeoxyribonucleotides, Antisense; Receptors, Notch; Repressor Proteins; Signal Transduction; Transcription Factors; Tyrosine 3-Monooxygenase; Ubiquitin-Protein Ligases; Zebrafish; Zebrafish Proteins