Methods for discussion Papers: Pasterkamp, R.J. et al “Sempaphorin 7A promotes axon outgrowth through integrins and MAPKs” Nature424: Fricke, C. “astray, a Zebrafish roundabout Homolog Required for Retinal Axon Guidance” Science 292:

Olfactory System Organization

Diagram of the mosaic olfactory projection in the main olfactory bulb (left) and accessory olfactory bulb (right). Drawing by Adam C. Puche, copyright Academic Press (Shipley, M.T., Puche, A.C., and Ennis, M. (2003) The olfactory system. In The Rat Nervous System. George Paxinos Ed., Academic press, NY). OE Olfactory Epithelium ONL Olfactory Nerve Layer GL Glomerular Layer EPL External Plexiform Layer MCL Mitral Cell Layer IPL Internal Plexiform Layer

Circuit diagram showing all major and minor cell types present in the main olfactory bulb. Drawing by Adam C. Puche, modified from Academic Press (Shipley, M.T., Puche, A.C., and Ennis, M. (2003) The olfactory system. In The Rat Nervous System. George Paxinos Ed., Academic press, NY). ONL Olfactory Nerve Layer GL Glomerular Layer EPL External Plexiform Layer MCL Mitral Cell Layer IPL Internal Plexiform Layer GCL Granule Cell Layer

Circuit diagram showing the major input and output circuits between the olfactory bulb, anterior olfactory nucleus and piriform cortex. Drawing by Adam C. Puche, copyright Academic Press (Shipley, M.T., and Puche, A.C. (2003) Cranial Nerve I: The Olfactory Nerve. In the Encyclopedia of Neurological Sciences, Academic Press, CA). LOT Lateral Olfactory tract AON Anterior Olfactory Nucleus PC Piriform Cortex

Some Components of Robo-like Receptors Ig Domains Fibronectin Type III repeats

Ig Superfamily Evidence for Ig domain 400 million years ago (vertebrates and invertebrates diverged) Superfamily includes many cell-surface proteins mediating cell-cell recognition (like N-CAM) Ig domains: amino acids long and thought to be folded into a sandwichlike structure made of two antiparallel β sheets, usually stabilized by a conserved disulfide bond. Alberts, 3 rd edition

Figure Schematic drawing of four forms of N-CAM. The extracellular part of the polypeptide chain in each case is folded into five immunoglobulinlike domains (and one or two other domains called fibronectin type III repeats for reasons that will become clear later). Disulfide bonds (shown in red) connect the ends of each loop forming each Ig- like domain (Alberts, 3 rd edition)

Fibronectin Large non-collagen adhesive protein Found in extracellular matrix Common to all vertebrates Multiple functional domains in subunits, most common is Fibronectin type III repeat Fibronectin type III repeat is 90 aa, occurs at least 15 times in each subunit Specific 3-peptide sequence of type III repeats is involved in cell binding– other factor determine specificity 0&WebEnv=0NduEOb1FVwxw5R9HYff2RhmAF1Uio93pgnAIH7uInmeLs9XLU9sAz&WebEnvR q=1&rid=cell.section.5140#5179

Genetic Mapping

Define Terms: CentiMorgan: (cM) The unit of linkage that refers to the distance between two gene loci determined by the frequency with which recombination occurs between them. Two loci are said to be one centimorgan apart if recombination is observed between them in 1% of meioses. Definition from: GeneTests from the University of Washington and Children's Health System, SeattleGeneTests About 0.74 megabase in zebrafish and 1 megabase in humans Shimoda et al (1999) Genomics 58: CentiRay: (cR) Refers to the size of the DNA fragments derived from treatment with a given dose of radition (cR 3000 for 3000 rad). If two genes are 1 centiRay apart (at a given dose of radiation), then there is a 1% chance of getting a break between them. In the zebrafish radiation hybrid map, one cR3000 is 61 kilobases. Geisler et al 1999 Nature Genetics 23:

Genetic Mapping with Microsatellite Markers Genetic Mapping attempts to determine position of a gene or marker within a chromosome This technique uses repetitive di- tri- and tetranucleotide sequences that occur frequently in the genome as markers They are called simple sequence length polymorphisms (SSLPs), simple sequence repeats (SSRs), or microsatellite markers CA repeats are the most common They are useful as markers because they are: Highly polymorphic (lots of alleles), widely dispersed, easily assayed by PCR, and applicable to diploid crosses The frequency of a meiotic cross-over between two genes is taken as a measure of how closely they are located. The closer they are, the less likely they are to cross over Shimoda et al 1999 Genomics 58: , WebEnv=0vIGjGa-_2oDAe4MaYkmZfkBVSeYjGjS05u44l1Hw0v-InCWyBA0AS&WebEnvRq=1&rid=mga.section.1855#1889

Radiation Hybrid Mapping Uses radiation of a certain dose to lethally irradiate cells and break the DNA, then fuses these cells to a rodent cell line Fragments are integrated into the rodent chromosomes or retained as “minichromosomes” The breaks can then be used for mapping in a manner similar to genetic mapping Resolution is higher, polymorphisms are unnecessary Identification of similar markers in RH and genetic maps allows the researcher to line up the maps and decipher more detailed information about the chromosome Geisler et al (1999) Nature Genetics 23:86-89