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：
7752479
负责人：
SEEMA AGARWALA
金额：
$ 31.7万
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 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7752479
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.

我们对脊椎动物细胞命运和回路组织的分子机制知之甚少中脑。中脑含有重要的核团（多巴胺能核、动眼核和红核），它们控制成瘾行为、眼睛、肢体和随意运动。我已经证明，在发育过程中，这些细胞核从指定的细胞命运（弧）条纹中出现来自 Sonic Hedgehog (SHH) 的消息来源。但我最近的发现提出了一个尚未探索的假设：SHH 虽然足够了，但对于中脑规范来说不是必需的，并且多个刺猬（SHH。印第安人，沙漠）源自神经（小鸡）或额外的神经源（小鼠、小鸡）可以共同塑造腹侧中脑。多配体 HH 信号如何转导产生特定的中脑细胞命运？我在 HH 配体（目标 1 和 2）以及最终和重要效应子的水平上检查这个问题。 HHcascade：Gli 基因（目标 3）。 Gli 1,2 和 3 在中脑中雕刻出 3 个离散的祖细胞域，可能反映一种可以指定不同中脑细胞命运的机制。尽管 Shh:Gli 基因相互作用已在脊髓，这些研究没有阐明中脑核团（例如多巴胺能）发育的重要性。该提案的ainfo 因此，我们的目标是研究多个 HH 和多个 Gli 基因在指定中脑细胞命运方面的相互作用。第一个目标是通过印度 shh-/- 的基因表达分析来探索单个 HH 基因和所有 HH 信号传导的作用。 Hedgehog (ihh-/-)、shh:ihh-l- 和平滑 (smo-/-) 小鼠（所有 HH 信号均丢失）。第二个目标将决定是否 SHH 是必要的，或者如果其他 HH 基因参与小鸡和小鼠急性功能丧失实验中的中脑模式。所有 HH 基因功能将被电穿孔 HH 信号传导负调控因子（PtcAloop2、HHIP）阻断。接下来个人Hh 通过电穿孔 RNA 干扰 (RNAi) 构建体，可以阻断小鸡和小鼠的基因功能。第三个目标将检查 Gli 基因在中脑模式形成中的作用。 Gli 基因将在离散的环境中过度表达并敲低 (RNAi) 由 GLI 基因表达描绘的中脑区域。 SHH-GLI 基因相互作用将在 shh-/-、smo-/- 和 g//3 -/- 中进行分析基因表达的单敲除和双敲除以及HH途径基因无机型的错误表达和敲低外植体。最后，小鸡 Talpid2 突变体中的中脑和脊髓缺陷，其中 SHH:GLI3 比率和侧底板规范受到干扰，将以基因表达为特征。将尝试通过以下方式拯救Ta2突变体：候选 Hh 通路基因（SHH、GLI3、DZIP1、HHIP；PTC1、SCUBE2）的错误表达。意义：HH-GLI 的研究通路对于理解中脑神经元的细胞命运和电路如何控制运动和成瘾至关重要行为，已被确定。了解中脑神经元的规格对于基于干细胞的开发也至关重要对抗帕金森病和中风的治疗策略。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A novel role for FOXA2 and SHH in organizing midbrain signaling centers.

DOI：
10.1016/j.ydbio.2012.06.018
发表时间：
2012-09-01
期刊：
DEVELOPMENTAL BIOLOGY
影响因子：
2.7
作者：
Bayly, Roy D.;Brown, Charmaine Y.;Agarwala, Seema
通讯作者：
Agarwala, Seema

Bone morphogenetic proteins regulate hinge point formation during neural tube closure by dynamic modulation of apicobasal polarity.

DOI：
10.1002/bdra.23052
发表时间：
2012-10
期刊：
BIRTH DEFECTS RESEARCH PART A-CLINICAL AND MOLECULAR TERATOLOGY
影响因子：
0
作者：
Eom, Dae Seok;Amarnath, Smita;Fogel, Jennifer L.;Agarwala, Seema
通讯作者：
Agarwala, Seema

A simple technique for early in vivo electroporation of E1 chick embryos.

DOI：
10.1002/dvdy.23747
发表时间：
2012-03
期刊：
Developmental dynamics : an official publication of the American Association of Anatomists
影响因子：
0
作者：
Brown CY;Eom DS;Amarnath S;Agarwala S
通讯作者：
Agarwala S

Apicobasal polarity and neural tube closure.