RESUMO
The control of growth, patterning, and differentiation of the mammalian forebrain has a large genetic component, and many human disease loci associated with cortical malformations have been identified. To further understand the genes involved in controlling neural development, we have performed a forward genetic screen in the mouse (Mus musculus) using ENU mutagenesis. We report the results from our ENU screen in which we biased our ascertainment toward mutations affecting neurodevelopment. Our screen had three components: a careful morphological and histological examination of forebrain structure, the inclusion of a retinoic acid response element-lacZ reporter transgene to highlight patterning of the brain, and the use of a genetically sensitizing locus, Lis1/Pafah1b1, to predispose animals to neurodevelopmental defects. We recovered and mapped eight monogenic mutations, seven of which affect neurodevelopment. We have evidence for a causal gene in four of the eight mutations. We describe in detail two of these: a mutation in the planar cell polarity gene scribbled homolog (Drosophila) (Scrib) and a mutation in caspase-3 (Casp3). We find that refining ENU mutagenesis in these ways is an efficient experimental approach and that investigation of the developing mammalian nervous system using forward genetic experiments is highly productive.
Assuntos
Etilnitrosoureia/efeitos adversos , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética , Ensaios de Triagem em Larga Escala , Mutagênicos/efeitos adversos , Doenças do Sistema Nervoso/genética , Prosencéfalo/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Mapeamento Cromossômico , Cruzamentos Genéticos , Impressões Digitais de DNA , Etilnitrosoureia/administração & dosagem , Feminino , Genes Reporter , Heterozigoto , Homozigoto , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutagênicos/administração & dosagem , Mutação , Doenças do Sistema Nervoso/embriologia , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Prosencéfalo/citologia , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/embriologia , TransgenesRESUMO
Organizing centers in the developing brain provide an assortment of instructive patterning cues, including Sonic hedgehog (Shh). Here we characterize the forebrain phenotype caused by loss of Ttc21b, a gene we identified in an ENU mutagenesis screen as a novel ciliary gene required for retrograde intraflagellar transport. The Ttc21b mutant has defects in limb, eye and, most dramatically, brain development. We show that Shh signaling is elevated in the rostral portion of the mutant embryo, including in a domain in or near the zona limitans intrathalamica. We demonstrate here that ciliary defects seen in the Ttc21b mutant extend to the embryonic brain, adding forebrain development to the spectrum of tissues affected by defects in ciliary physiology. We show that development of the Ttc21b brain phenotype is modified by lowering levels of the Shh ligand, supporting our hypothesis that the abnormal patterning is a consequence of elevated Shh signaling. Finally, we evaluate Wnt signaling but do not find evidence that this plays a role in causing the perturbed neurodevelopmental phenotype we describe.
