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We have a number of ongoing projects and are always interested in discussing them further or integrating new lab members into these efforts.
We primarily use this forward genetic approach to identify genes not previously known to be required in development. We use the chemical mutagen ENU to create random mutations in the genome. We then breed potential mutations to homozygosity to create recessive developmental phenotypes. A combination of cutting-edge genomics and positional cloning is used to identify the mutated gene leading to the phenotypes. This turns out to be a relatively efficient tool for gene discovery in development. (see Stottmann et al., 2011; Stottmann and Beier 2010). We continue to use ENU mutagenesis to create novel mutations and will increasingly employ transgenic lines to more specifically query various aspects of neuronal specification and migration.
An ENU mutagenesis experiment designed to recover monogenic, recessive phenotypes. Half-shading indicates heterozygosity for a hypothetical ENU mutation and full-shading indicates homozygosity (modified from Stottmann et al., 2011, Genetics)
Examples of the brain and craniofacial malformations we have recovered to date, both in whole mount and in a histological preparation (frontal sections).
The alien mutation was identified in a mutagenesis screen in the lab of David Beier at the Brigham & Women's Hospital (Herron et al., Nat Genetics 2002). Positional cloning revealed this to be a null mutation in the gene Ttc21b/ Thm1,which is required for protein transport within the primary cilium (Tran et al., Nat Genetics 2008). The alien mutant also has a severe brain patterning defect (Stottmann et al., Dev Biol, 2009). The role of Ttc21b in the developing forebrain and craniofacial tissues, and the role of cilia in development more broadly, is a focus within the laboratory. One direction of this project is a tissue specific ablation of Ttc21b in the developing forebrain.
The alien forebrain has significant patterning defects at E18.5 (modified from Stottmann et al., Dev Biol 2009)
Emx1-Cre recombination pattern in the forebrain of the E13.5 embryo (eGFP Cre reporter).
Alien mutant embryos have craniofacial malformations.
The rudolph mutation was recovered in another ENU mutagenesis screen in the Beier lab (Stottmann et al., Genetics, 2011). Positional cloning identified an intronic mutation creating a hypomorphic allele of the gene hydroxysteroid (17-beta) dehydrogenase 7 (Hsd17b7; Stottmann et al., PLoS Genetics, 2011). Further studies of the rudolph mutation demonstrated for the first time in a genetic, in vivo, animal model a requirement for sterol metabolism in proper Sonic Hedgehog intracellular signal transduction. The rudolph mutants have a striking defect in tissue organization throughout the CNS which is not due to dysregulation of the Shh pathway. Current efforts in the lab are aimed at understanding the molecular mechanisms underlying this defect. This will contribute to a greater understanding of the role of cholesterol metabolism in cortical development.
Rudolph mutants at E16.5 show a number of phenotypes including an extremely disorganized cortex (modified from Stottmann et al., PLoS Genetics, 2011).
We are currently exploring ways to identify the genetic basis of human congenital malformations. We are using next-generation exome sequencing to study potential genetic variants in patients seen here at CCHMC. We are also trying to determine which human syndrome(s) are caused by mutations in the same genes we discover in our mouse experiments. To do this, we are pursuing a number of collaborations both within the Cincinnati Children's Hospital Medical Center, as well as at a number of other institutions around the world.
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