The goal of this proposal is to define the general principles of dorsal medial germinal zone organization and regulation, using Lmx1a as a molecular entry point. Dorsal brain germinal zones, such as the cerebellar rhombic lip and telencephalic cortical hem, contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive their neurogenesis are ill-defined. Using genetic fate mapping and mutant analysis we have demonstrated that during cerebellar development, the LIM-homeodomain transcription factor Lmx1a 1) segregates the roof plate lineage from neuronal rhombic lip derivatives, 2) is required for maintenance of the entire late embryonic rhombic lip and 3) confers posterior vermis identity to a subset of rhombic lip cells. These experiments demonstrate that the cerebellar rhombic lip is a heterogeneous progenitor population with fates specified at very early stages, yet we know nearly nothing about the cellular and molecular characteristics of rhombic lip progenitors.
In Aim 1 of this proposal we will conduct an extensive analysis of rhombic lip progenitor gene expression and cell cycle parameters in wild-type and Lmx1a-/- animals to characterize the organization, developmental mechanisms and molecular pathways conferring cell fate within the rhombic lip. Using expression microarray analysis of microdissected e13.5 cerebellar rhombic lip from wild-type and Lmx1a-/- animals, we have identified several Lmx1a-candidate effectors and we will characterize their roles in rhombic lip development through analysis of extant mouse mutants and manipulation of gene expression using in utero mouse electroporation. Our preliminary data demonstrate that Lmx1a also regulates neurogenesis in the telencephalic cortical hem, where loss of Lmx1a results in aberrant expression of the cortical selector gene Lhx2, which leads to excessive production of hippocampal cells instead of Cajal-Retzius cells.
In Aim 2 we propose a series of genetic fate mapping and in vitro explant analyses to test if Lmx1a acts intrinsically in the cortical hem to regulate cortical hem neurogenesis and if the adjacent telencephalic choroid plexus, where Lmx1a is also expressed, also influences hem development. We will also conduct expression analyses to determine if Lmx1a-downstream effectors are conserved across dorso-medial germinal zones. Finally in Aim 3, we will explore the basis of dorsal midline abnormalities when both Lmx1a and Lmx1b function removed. These basic neurodevelopmental analyses have direct translational relevance to human structural malformations of the CNS and have potential to reveal new insights into the dramatic evolution of cortical size in mammals.
With recent advances in imaging and genetics, malformations of the human brain are more frequently recognized. These malformations cause neurodevelopmental and motor delays in affected children, yet we know very little regarding which processes that go awry while the brain is developing or the underlying genetic causes. This grant uses genetic manipulations in the mouse to define the organizational principles of the embryonic stem cell populations that reside on the dorsal midline of the brain and generate vast numbers of neurons in the adult brain responsible for memory and motor coordination. When these stem cell populations develop abnormally, birth defects of the brain including cerebellar and cerebral malformations and hydrocephalus result. An improved understanding of their basic developmental biology will ultimately lead to improved diagnosis and eventually treatment for these devastating brain disorders.
|Mirzaa, Ghayda M; Millen, Kathleen J; Barkovich, A James et al. (2014) The Developmental Brain Disorders Database (DBDB): a curated neurogenetics knowledge base with clinical and research applications. Am J Med Genet A 164A:1503-11|
|Millen, Kathleen J; Steshina, Ekaterina Y; Iskusnykh, Igor Y et al. (2014) Transformation of the cerebellum into more ventral brainstem fates causes cerebellar agenesis in the absence of Ptf1a function. Proc Natl Acad Sci U S A 111:E1777-86|
|Sillitoe, Roy V; George-Jones, Nicholas A; Millen, Kathleen J et al. (2014) Purkinje cell compartmentalization in the cerebellum of the spontaneous mutant mouse dreher. Brain Struct Funct 219:35-47|
|Doherty, Dan; Millen, Kathleen J; Barkovich, A James (2013) Midbrain and hindbrain malformations: advances in clinical diagnosis, imaging, and genetics. Lancet Neurol 12:381-93|