Lecture1note(1).pdf

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Karina S. [email protected]

9/26/13!

Developmental Neurobiology!N152!

N152 : Course Information!Class meets: RH 104, Tu Thu 8:00-9:20a.m.!Final Exam: Thursday, December 10, 8:00 - 10:00 a.m.!!Professors: !!

Dr. Karina S. [email protected]!Office Hours: Tuesday 1pm, room 2246 MH!!Dr. Susana [email protected]!Office Hours: Tuesday 1pm, room 2246 MH!!

Teaching Assistant:!!Emily Vogler [email protected]!Office Hours: TBA!!


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N152 : Course Information!Textbook:!

Development of the Nervous System by Sanes, Reh,

and Harris, 3rd edition!!Class Web Site: ! https://eee.uci.edu/13f/06211!!Grading:!

Midterm Exam (October 29, 2013) !40%!Final Exam (December 10, 2013) !40%!Online quizzes ! ! !10%!Neuroscience assignment ! !10%!!

What is Developmental Neurobiology?!The study of how the nervous system is assembled duringembryonic development and throughout later stages of life! Takes into account anatomical and physiological changes,and focus is on cellular and molecular mechanisms.! Considers specializations, but also seeks to determinerules for developmental processes, therefore requiresexamination of multiple systems.! Must start with basic knowledge about mature nervoussystem organization and function.!


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Course Goals

What are the morphological events that lead to the formation ofthe mature nervous system?!

How do we find out about these events?! What are the cellular and molecular events that lead to mature

morphology and connectivity?! What are the general kindsof molecular interactions?

Conserved across species, reused during different stages of

development.! How do we test the roles of individual molecules?!

Fun Facts about the Brain

Usually 1 brain/person, weight about 2.5 lbs.!100,000 miles of axons/brain, conduct 200 MPH!Brain uses 20% of body's blood flow and energy!25% of human genes are expressed in the brain!About 1,000,000,000,000 (1012) neurons/brain!

- Each with thousands of inputs, up to 150,000!- Each separated from all others by 6 deg separation!- Approximately 20 glial cells/neuron!- Diameter = 8 - 80 m !


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How do cells acquire their neuronal identity?!Differential gene expression and signals that introduce differences

between initially identical cells!

How do you achieve synaptic specificity?!Tightly controlled developmental processes!

How does the nervous system develop?!

?!


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Figure 22-1 Molecular Biology of the Cell( Garland Science 2008)

Essential processes that make a

multicellular organism!

and cell death.!

How do we study nervous systemdevelopment?!

Model Systems!Experimental approaches !


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Vertebrate models to study development!

Vertebrate models to study development!FrogsXenopuslaevis

FishZebrafish(Daniorerio)

Chick

Mouse

Thebasicmachineryofdevelopmentisthesamenotjustinall

vertebratesbutinallthemajorphylaofinvertebrates


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Frogs Xenopus laevis!

Adults are easily bred and maintained in laboratory!Embryos develop externally (outside of mother)!Can be viewed and manipulated throughout development!Individual cells can be labeled with lineage tracers - fate mapping!Cells and tissues can be ablated or transplanted easily!Easy to overexpress genes by RNA injection!

Frogs Xenopus laevis!Egg to tadpole in 110hrs!

Judith Cebra-Thomas: !http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Frog/frog_staging.html!Figure 22-67 Molecular Biology of the Cell( Garland Science 2008)


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Zebrafish - (Danio rerio) !

Adults are easily bred and maintained in laboratory!Embryos develop externally (outside of mother)!Can be viewed and manipulated throughout development!Mutagenesis and genetic screens!Cells and tissues can be ablated or transplanted easily!Easy to create transgenics!

Development can be studied in ovoor in whole embryo culture!Morphogenetic studies growth of cells and tissues!Fate mapping by dye tracing or transplantation (chick-quail chimeras)!Viral vectors (retrovirus) used for gene expression!Electroporation of foreign genes!

Chick!


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Advantages! Genetic technologies: transgenics, KOs, conditionals (cre/loxp)! Greater genetic homology with humans (mammalian)! Whole embryo culture few days! Fetal and subsequent postnatal development mimics humans better!Disadvantages!Develop inside uterus !Opaque!Longer gestation!

Mouse!

Figure 22-24 Molecular Biology of the Cell( Garland Science 2008)!

Fruit fly: Drosophila melanogaster!

Invertebrate systems widely used !in developmental neurobiology!


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Mutagenesis and !Genetic screens!(reproduce rapidly)!!



Nematode worm:!C. Elegans!!

Cell lineage is stereotyped!Development takes only 3 days!Mutagenesis and genetic screens!Can knockdown genes with siRNA!



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Methods in developmental neurobiology!Description!

Time course of events!Correlations with other events!

Perturbations: Gain and loss of function!!Add or remove tissues, factors, genes!Test effect on nervous system development!

Methods in developmental neurobiology!Mutagenesis !Removing or transplanting tissue!Cell ablation!Gene deletion: KO or conditional KO!Cell lineage tracing; fate mapping!


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Methods in developmental neurobiology!Mutagenesis !Removing or transplanting tissue!Cell ablation!Gene deletion: KO or conditional KO!Cell lineage tracing; fate mapping!

Mutagenesis!Generates random mutations in genes. Typically done in flies,worms, and zebrafish.!Chemical mutagen!Radiation!Insertional mutagenesis: !!Random insertion of an exogenous DNA sequence that can be easily

detected so that you can figure out which gene it inserted into andcorrelate that with a given phenotype.!


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Mutagenesis!

Groups of genes that allcause a very similar

phenotype are often part of

the same molecular pathway.!

Early drosophila development screen reveals egg polarity genes!!

Mutagenesis screens!

Tudor A. Fulga et al., Nature Cell Biology9, 139 - 148 (2007)!

The Rough Eye phenotype provides a simple indication of a

problem with the CNS!


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Figure 22-2a Molecular Biology of the Cell( Garland Science 2008)

Homologous proteins can function

interchangeably in humans, mice, and flies!

Cerebellum doesnt develop!

Figure 22-2b Molecular Biology of the Cell( Garland Science 2008)

Squid !Pax 6!Expressed !In the leg!!

Drosophila!Eyeless!Expressed !In the leg!!

Normal!Eye!

Homologous proteins can function

interchangeably in humans, mice, and flies!


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Hox Genes!

Influence anterior-posteriorpatterning across phyla -- in

both flies and humans !!

Mutagenesis screens!

(A) Neuromast from a control animal stained with FM 1-43FX (red) and the nuclear label Yo-Pro-1 (green). (B) Negative control pretreated with0.05% DMSO for 1 hour followed by 200 M neomycin treatment for 30 min. Hair cells are stained with FM 1-43FX (red) and Yo-Pro-1 (green). Hair

cell loss, nuclear condensation and cytoplasmic shrinking are observed. (C) Dose-response function showing decreased hair cell labeling withDASPEI, a mitochondrial potentiometric dye, as a f unction of increasing neomycin concentration for wildtype zebrafish Screens for increased ordecreased susceptibility to hair cell loss were performed by treatment with either low, 25 M, or high, 200 M, neomycin doses, respectively, ashighlighted by the orange arrows. (D) Neuromast pretreated with PROTO-2, a compound identified to provide protection against 200 M neomycinexposure. From Owens et al., PLoS Genetics vol4(2), Feb 2008.!

Example: Protection from or vulnerability to aminoglycosides!!


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Methods in developmental neurobiology!Mutagenesis !!Removing or transplanting tissue!Cell ablation!Gene deletion: KO or conditional KO!Cell lineage tracing; fate mapping!

Tests for: !!The presence of potential inducers in a particular tissue, cell ororgan!The ability of tissues and/or cells to respond to inductive signals!The timing of neural specification or commitment to a particularcell fate. !

Removing and transplanting tissue!


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Tissue transplantation!!isochronic (same developmental stage)!!heterochronic (different developmental stage)!!homotopic (donor-to-host identical site)!!heterotopic (donor-to-host ectopic site)!Ectopic or over-expression of molecules!!transgenics (constitutive gene expression)!!cDNA transfection (microinjection, electroporation, virus infection)!

!protein diffusion (implantation of beads, COS cells)!

Cellular level!

Molecular equivalent!

Removing and transplanting tissue!

Figure 22-6b Molecular Biology of the Cell( Garland Science 2008)

Transplantation of tissue from a pigmented

embryo into a non-pigmented host induces asecond axis and a secondary embryo!


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Tissue transplants to examine whether a

cells fate is determined!


The thigh tissue has alreadybeen programmed to become

leg-associated tissue, but hasnot yet been specified to

become thigh vs. toes.!


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Allows to determine if an organ, tissue or cell, or even a

molecule is necessary for a particular event in development.!Cellular level!Tissue ablation!Cell ablation!! (laser ablation)!Tissue or cell isolation!! (explant or cell cultures to test cell-fate specification)!!Molecular equivalent!Gene knockdown or knockout!Protein downregulation (RNAi, microRNAs)!!!

Modulate gene expression and ablation

of specific cell populations !


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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.!

Adaption of Cre-Lox technique to ablate new-born neurons



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Only new-born neurons are killed in vivo!




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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!

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 specific promoter/enhancer reporter gene expression

(Cre/loxP-GFP)!


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Cell Lineage Tracing (fate mapping)!

Injection of 2 different vital dyesinto early chick embryo. Later

the progeny of these regionscan be seen.!

Injection of 3 different vital dyes into early Xenopus embryo. These studies

reveal the normal fates of the progeny of individual cells.!



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Nature Cell Biology3, 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 shouldeliminate neural crest, neural crest cells still emerge. Theresults 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 fora few hours after the time of normal onset of emigration from the neural tube.!

GFP transgenics with developmentally-regulated Cre expression togive temporal specificity!

Drosophila!Xenopusnervous system!

Mouse embryos! Retina photoreceptors!

Molecular genetic tools for studying cell lineage!


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Genetic inducible fate mapping!Cre lines allow identification of the fatesof cells that expressed a given gene

(here, wnt1). !Inducible cre lines allow analysis of thefates of cells at different times. !These spatially and temporally limitedmutations can be useful in determining

which cells lead to a particular fate,

during normal development, after aperturbation, or in disease models.!Can also use Cre-loxP and Flp-FRT,alone or in combination.!

J. Vis. Exp., 2009 Dec 30;(34)

Summary!Neural development involves proliferation, differentiation,cellular communication, and cell migration.!Our knowledge of neural development comes from avariety of model organisms, each of which has distinctadvantages that make them ideal for certain experiments.!Experiments that reveal cellular and molecularmechanisms of development involve removalor additionoftissues or molecules, and analysis of the effects ondevelopment.!Diverse species share some molecular mechanismsduring development.!

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