Molecular Mechanisms in Vertebrate Midbrain Development

脊椎动物中脑发育的分子机制

基本信息

批准号：
7782074
负责人：
SEEMA AGARWALA
金额：
$ 2万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7782074
关键词：
Acute Addictive Behavior Affinity Animal Model Biological Assay Brain Candidate Disease Gene Cell Death Cell Nucleus Cell Proliferation Cells Clinical Complex Data Defect Development Electroporation Erinaceidae Eye Floor GLI gene GLI2 gene GLI3 gene Gene Expression Gene Family Gene Proteins Genes Goals In Vitro Individual Knockout Mice Lateral Ligands Limb structure Mammals Midbrain structure Molecular Motor Movement Mus Mutant Strains Mice Neural tube Neurons PTCH gene Parkinson Disease Pathway interactions Pattern Pattern Formation Process RNA Interference Red nucleus structure Research Personnel Role Signal Transduction Signaling Molecule Source Specific qualifier value Spinal Cord Stem Cell Development Stroke Substantia nigra structure Testing Therapeutic Ventral Tegmental Area Zebrafish base cell fate specification cell type combat gene function gene interaction hedgehog signal transduction human SMO protein in vivo knock-down knockout gene loss of function mutant oculomotor overexpression progenitor programs protein expression relating to nervous system research study transcription factor

项目摘要

We know very little about the molecular mechanisms underlying the organization of cell-fates and circuitry in the vertebrate midbrain. The midbrain contains important nuclei (dopaminergic, oculomotor and red nuclei) that govern addictive behaviors, eye, limb and voluntary movements. I have shown that during development, these nuclei emerge from stripes of cell-fates (arcs) specified by a source of Sonic Hedgehog (SHH). But my recent findings suggest an as yet unexplored hypothesis: that though sufficient, SHH is not necessary for midbrain specification and that multiple hedgehogs (SHH. Indian, Desert) derived from neural (chick) or extra- neural sources (mouse, chick) could jointly pattern the ventral midbrain. How is the multi-ligand HH signal transduced to produce specific midbrain cell-fates? Iexamine this issue at the level of the HH ligands (Aims 1and 2) and the final and essential effectors of the HHcascade: the Gli genes (Aim 3). Gli 1,2 and 3 carve out 3 discrete progenitor domains in the midbrain, possibly reflecting a mechanism by which distinct midbrain cell-fates could be specified. Although Shh:Gli gene interactions have been studied in the spinal cord, these studies do not elucidate how important midbrain nuclei (e.g. dopaminergic) develop. The ainfof this proposal therefore is to investigate the interactions between multiple HH and multiple Gli genes in specifying midbrain cell-fates. The first aim will explore the role of individual HHgenes and all HH signaling by gene expression analyses of the shh-/-, Indian Hedgehog (ihh-/-), shh:ihh-l- and smoothened (smo-/-) mice (where all HHsignal is lost). The second aim will determine whether SHH is necessary or if other HHgenes are involved in midbrain patterning in acute loss of function experiments in chicks and mice. All HH gene function will be blocked by electroporating negative regulators of HH signaling (PtcAloop2, HHIP). Next, individual Hh gene function will be blocked in chicks and mice by electroporation of RNA interference (RNAi) constructs. The thirdaim will examine the role of Gli genes in midbrain patterning. Gli genes will be overexpressed and knocked down (RNAi) in the discrete midbrain domains delineated by GLI gene expression. SHH-GLI gene interactions will be analyzed in the shh-/-, smo-/- and g//3 -/- single and double knockouts by gene expression and the misexpression and knockdown of HH pathway genes inorganotypic explants. Finally, midbrain and spinal cord defects in the chick Talpid2 mutant, where the SHH:GLI3 ratio and lateral floor plate specification are perturbed, will be characterized by gene expression. Attempts will be made to rescue the Ta2 mutant by misexpression of candidate Hh pathway genes (SHH, GLI3, DZIP1, HHIP; PTC1, SCUBE2). Significance: A study of the HH-GLI pathway is critical for understanding how the cell-fates and circuitry of midbrain neurons, which govern motor and addictive behaviors, are determined. Understanding the specification of midbrain neurons is also crucial to the development of stem cell based therapeutic strategies to combat Parkinson's disease and stroke.