Molecular Control of Neural Cell Fate Determination

神经细胞命运决定的分子控制

• 批准号: 7611994
• 负责人: David J Anderson
• 金额: $ 34.21万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7611994
• 关键词: Address; Algorithms; Astrocytes; Biological Assay; Candidate Disease Gene; Categories; Cell Communication; Cells; Cellular biology; Chick Embryo; Classification; Code; Competence; Data; Development; Electroporation; Embryo; Event; Exhibits; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Expression Profiling; Genes; In Situ Hybridization; In Vitro; Interneurons; Kinetics; Knock-out; Length; Maps; Methods; Molecular; Motor Neurons; Mus; Neuroglia; Neurons; Oligodendroglia; Oligonucleotide Microarrays; Oligonucleotides; Pattern; Play; Population; Process; Property; RNA Interference; Regulation; Regulator Genes; Reporter; Repression; Reverse Transcriptase Polymerase Chain Reaction; Role; Series; Spinal Cord; Staging; Stem cells; System; Techniques; Testing; Time; Transcription Repressor/Corepressor; Transplantation; Ventricular; base; comparative; gain of function; gene interaction; genetic manipulation; gliogenesis; in vivo; loss of function; molecular marker; mutant; nervous system disorder; neurodevelopment; neurogenesis; progenitor; relating to nervous system; research study; self-renewal; stem; transcription factor

The objective of this proposal is to understand the cellular and molecular mechanisms that control a fundamental event in neural development: the switch from neurogenesis to gliogenesis. An understanding of this process is essential for applying neural stern and progenitor cell biology to the treatment of neurological disease. This switch will be studied in a specific population of spinal cord precursors that sequentially generate motoneurons (MNs) and oligodendracytes (oligos). These precursors can be prospectively isolated using fluorescence-activated cell sorting (FACS), by means of a GFP reporter expressed from the Olig2 locus, which encodes a transcription factor required for both MN and oligo differentiation. Using these isolated cells, we will address the following specific aims: I). We will test whether MNs and oligos develop from a multipotential, self-renewing stem cell in the ventricular zone (VZ) of the spinal cord, as is widely assumed, or rather from progenitors that undergo irreversible restrictions in developmental competence. We will investigate this by using a newly developed technique for direct transplantation of freshly isolated Olig2-expressing progenitors into the chick spinal cord, without any ex vivo expansion (which may perturb the properties of the cells). Using this approach, we will perform heterochronic transplantation experiments to test the self-renewal anc developmental capacities of Olig2-i- cells at different stages during the MNgoligo transition. II). We will test the hypothesis that changes in gene expression in Olig2+ progenitors during the neuron-to-glial switch reflect the regulation of several distinct subclasses of genes, each with different kinetics of activation and repression. This hypothesis will be tested by using oligonucleotide microarrays to perform gene expression profiling (GEP) experiments on acutely isolated Olig2+ progenitors from different stages of spinal cord development. This GEP analysis should also identify a) markers useful in clarifying lineage relationships between Olig2+ progenitors of MNs and oligos; and b) candidate regulatory genes for functional analysis. Ill) We will tost the hypothesis that there is a "temporal code" of transcription factors that controls the MN->oligo switch, by performing gain-of-function (GOF) and loss-of-function (LOF) genetic manipulations of candidate regulatory genes identified in the GEP temporal analysis (Aim II). Electroporation of chick spinal cord will be used as a rapid in vivo assay for such functional manipulations, employing expression of full-length cDNAs, and independently validated shRNAi (small hairpin RNAi) constructs, for GOF and LOF experiments, respectively. The embryos will be analyzed uning an extensive battery of molecular markers for various classes of neurons (including MNs and interneurons), oligodendrocytes, and newly validated markers of astrocytes and their progenitors. Candidates for which strong functional data is obtained from the chick system will be further validated by generating constitutive or conditional knockouts in the mouse. IV) We will test the hypothesis that targets of OLIG2, which functions as a transcriptional repressor, include a) repressers of oligo differentiation; and b) activators of astrocyte differentiation. Candidate targets of OLIG2 will be identified by performing comparative GEP analysis of isolated Olig2-GFP+ cells from Olig1/2+/- and Olig2-/- spinal cord, at several developmental stages. A series of analytic algorithms will be used to filter the data to obtain a list of transcription factors that are de-repressed in the absence of OLIG2 function. These candidates will be further validated and prioritized by real-time RT-PCR and in situ hybridization. Top candidates will then be functionally analyzed by GOF and LOF manipulations in chick embryos.

该提案的目的是了解控制神经发育中基本事件的细胞和分子机制：从神经发生到神经胶质发生。对这一过程的理解对于将神经船尾和祖细胞生物学应用于神经系统疾病的治疗至关重要。该开关将在特定的脊髓前体群中进行研究，这些脊髓前体依次产生运动神经元（MNS）和寡头胶囊（Oligos）。这些前体可以通过荧光激活的细胞分选（FACS）前瞻性地分离，通过从Olig2基因座表达的GFP报告基因，该基因座编码MN和Oligo分化所需的转录因子。使用这些孤立的单元格，我们将解决以下特定目的：i）。我们将测试MN和寡核能是否从心室区域的多能自我更新干细胞发展 （Vz）的脊髓，正如广泛假定的，或者是来自在发育能力中受到不可逆转限制的祖细胞。我们将通过使用新开发的技术将新鲜分离的寡聚祖细胞直接移植到鸡脊髓中，而无需任何外体膨胀（这可能会扰乱细胞的性质）。 使用这种方法，我们将执行异缘人移植实验，以测试mngoligo跃迁期间不同阶段olig2-i-细胞的自我更新ANC发育能力。 ii）。我们将检验以下假设：基因表达的变化 神经元到胶质开关期间的Olig2+祖细胞反映了基因的几个不同亚类的调节，每个基因都具有不同的激活和抑制动力学。该假设将通过使用寡核苷酸微阵列在急性分离的Olig2+祖细胞上从脊髓发育的不同阶段进行基因表达分析（GEP）实验来检验。该GEP分析还应确定a）标记有助于阐明MNS和寡聚物的Olig2+祖细胞之间的谱系关系； b）用于功能分析的候选调节基因。我们将提出一个假设，即通过执行功能障碍（GOF）和功能丧失（LOF）遗传操纵在GEP时间分析中鉴定出的候选调节基因（AIM II）来控制Mn-> oligo开关的“时间代码”。雏鸡的电穿孔将用作 用于这种功能操纵的快速体内测定，分别采用全长cDNA的表达以及独立验证的shrnai（小发夹RNAi）构建体，分别用于GOF和LOF实验。将对各种神经元（包括MN和中间神经元），少突胶质细胞和 新近验证的星形胶质细胞及其祖细胞的标记。通过在小鼠中产生组成型或条件敲除，从雏鸡系统获得强功能数据的候选物将得到进一步验证。 iv）我们将检验以下假设 充当转录阻遏物的Olig2的靶标包括a）寡素分化的阻遏物； b）星形胶质细胞分化的激活剂。在几个发育阶段，将通过对Olig1/2 +/-和Olig2 - / - 脊髓进行分离的Olig2-GFP+细胞的比较GEP分析来鉴定Olig2的候选靶标。一系列的分析算法将用于过滤数据，以获取在没有Olig2函数的情况下取消重新定义的转录因子列表。这些候选人将通过实时RT-PCR和原位杂交对这些候选人进行进一步验证和优先级。然后，最高候选人将通过功能分析 小鸡胚胎中的GOF和LOF操纵。

项目成果

Sympathetic neuroblasts undergo a developmental switch in trophic dependence.

交感神经母细胞经历营养依赖性的发育转变。

• DOI: 10.1242/dev.119.3.597
• 发表时间: 1993
• 期刊: Development (Cambridge, England)
• 作者: Birren,SJ;Lo,L;Anderson,DJ
• 通讯作者: Anderson,DJ

Co-expression of multiple neurotransmitter enzyme genes in normal and immortalized sympathoadrenal progenitor cells.

正常和永生化交感肾上腺祖细胞中多种神经递质酶基因的共表达。

• DOI: 10.1016/0012-1606(91)90313-r
• 发表时间: 1991
• 期刊: Developmental biology
• 影响因子: 2.7
• 作者: Vandenbergh,DJ;Mori,N;Anderson,DJ
• 通讯作者: Anderson,DJ

MASH genes and the logic of neural crest cell lineage diversification.

MASH 基因和神经嵴细胞谱系多样化的逻辑。

• 发表时间: 1993
• 期刊: Comptes rendus de l'Academie des sciences. Serie III, Sciences de la vie
• 作者: Anderson,DJ

Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons.

发育交感神经元中转录因子基因表达和表型标记的差异调节。

• DOI: 10.1242/dev.121.3.887
• 发表时间: 1995
• 期刊: Development (Cambridge, England)
• 作者: Groves,AK;George,KM;Tissier-Seta,JP;Engel,JD;Brunet,JF;Anderson,DJ
• 通讯作者: Anderson,DJ

Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo.

• DOI: 10.1038/nature11810
• 发表时间: 2013-01-31
• 期刊: Nature
• 影响因子: 64.8