Lecture2NotesRevised

Lecture2NotesRevised - Developmental Neurobiology! N152!...

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1 Karina S. Cramer cramerk@uci.edu 9/27/11 Developmental Neurobiology N152 Cre Recombinase – Lox P Transgenics (Imayoshi et al., Nature Neuroscience, 2008) Allows for genetic control of limited populations of cells, determined by the promoter that drives expression of cre .
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2 Adaption of Cre-Lox technique to ablate new-born neurons (Imayoshi et al., Nature Neuroscience, 2008) Only new-born neurons are killed in vivo (Imayoshi et al., Nature Neuroscience, 2008)
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3 Methods in developmental neurobiology Mutagenesis Removing or transplanting tissue Cell ablation Gene deletion: KO or conditional KO Cell lineage tracing; fate mapping Cell lineage tracing and fate mapping Cell lineage tracing : determine which cells and cell divisions give rise to cells or groups of cells Fate mapping : determine what a particular cell or group of cells will give rise to later in development
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4 Cell lineage tracing and fate mapping Cellular/Tissue approaches Generation of chimeric embryos by tissue transplantation (amphibians, chick-quail chimeras) Vital tracer dyes (microinjection into tissues and cells) Antibody staining (based on cellular phenotype) Molecular tagging Epitope tag (e.g., lacZ – antibody detection) Fluorescent proteins (GFP, DsRed) Tissue speci±c promoter/enhancer reporter gene expression (Cre/loxP-GFP) Figure 22-5 Molecular Biology of the Cell (© Garland Science 2008) Cell Lineage Tracing (fate mapping) Injection of 2 different vital dyes into early chick embryo. Later the progeny of these regions can be seen.
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5 Injection of 3 different vital dyes into early Xenopus embryo. These studies reveal the normal fates of the progeny of individual cells. Cell Lineage Tracing (fate mapping) Cell Lineage Tracing (fate mapping) Nature Cell Biology 3 , E216 - E218 (2001) Double-labelling with 'DiO' and 'DiI' illustrate that ablated regions and not the unoperated neural folds form the neural crest after ablation. After a lesion that should eliminate neural crest, neural crest cells still emerge. The results of this fate mapping study show that neural tube cells (normally destined to form CNS) can change their fates to form PNS and other neural crest derivatives for a few hours after the time of normal onset of emigration from the neural tube.
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6 GFP transgenics with developmentally-regulated Cre expression to give temporal specifcity Drosophila Xenopus nervous system Mouse embryos Retina photoreceptors Molecular genetic tools ±or studying cell lineage Genetic inducible ±ate mapping • Cre lines allow identifcation o± the ±ates o± cells that expressed a given gene (here, wnt1). • Inducible cre lines allow analysis o± the ±ates o± cells at di±±erent times. • These spatially and temporally limited
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Lecture2NotesRevised - Developmental Neurobiology! N152!...

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