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We are currently collaborating with our clinical partners to directly study patients with congenital malformations of the brain and face.
We are actively enrolling in research protocols in each of these areas and using whole genome sequencing to identify potentially causal genetic variants in these patients. We then employ CRISPR / Cas9 genome editing, molecular embryology, animal models, and induced pluripotent stem cells (IPSCs) to try and understand how these variants lead to malformations.
As part of this work, we are pursuing a number of collaborations within Cincinnati Children's, as well as at a number of other institutions around the world. This is an area of tremendous growth and opportunity as we continue to enroll patients and perform research sequencing. The complementary tools of genome sequencing, CRISPR genome editing, animal models and stem cell technologies offer almost limitless chances for discovery.
Work on this project is funded by the NIH (NINDS and NIDCR) and the Cincinnati Children's Research Foundation Center for Pediatric Genomics.
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.
Work on this project is funded by the Cincinnati Children's Research Foundation and the NIH (NINDS).
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).
Mouse ENU mutants with craniofacial abnormalities.
The alien mutation was previously identified in a mutagenesis screen (Herron et al., Nat Genetics 2002). Positional cloning revealed this to be a null mutation in the gene Ttc21b, 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 major focus within the laboratory. One direction of this project is a tissue specific ablation of Ttc21b and other primary cilia genes in the in the developing forebrain and craniofacial tissues.
Evidence from human genetics studies suggest TTC21B is a node in a larger ciliary signaling network. We are studying how these genes interact through studies in the mouse and in vitro. We are also using a forward genetic approach to identify novel loci interacting with Ttc21b in mammalian development and disease. We have also used a Quantitative Trait Locus analysis to identify a gene we now believe at least partially explains the effects of genetic background on the alien forebrain mutation.
Work on this project is funded by the Cincinnati Children's Research Foundation, the March of Dimes Foundation and the NIH (NIGMS).
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.
Tubulin proteins are the building blocks of microtubules, a key element of the cellular cytoskeleton. We and others have shown in mouse and humans that variants in tubulins lead to drastic malformations of cortical development. The tubulin genes together make up a family of genes with high-sequence similarity and, often, adjacent positions in the genome. We are using genome editing techniques to make a series of alleles to test the requirements for these genes as this has never been tested. We are also generating new models of human disease variants and constructing a new tool to address expression and regulation of tubulin genes.
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).