Cellular, molecular and genetic analysis of neural fate in Zebrafish embryos

我们的目标是确定细胞、分子和遗传机制，这些机制决定了神经元如何在神经板中形成适当的数量和位置。为了确定这种机制，我们已经确定了斑马鱼突变体具有异常模式的早期神经元。对这些突变体的分析开始确定斑马鱼胚胎早期神经发生模式所必需的机制。无头（hdl）突变体通过其扩大的三叉神经结构域进行鉴定。在该突变体中，三叉神经元的扩张伴随着中脑-后脑边界（MHB）神经域的扩张，而三叉神经元通常与中脑-后脑边界相邻。与MHB相关的神经区域的扩张是以眼睛、前脑和大部分中脑等前神经结构为代价的，这些结构在这种突变中丢失了。在早期原肠胚形成过程中，Wnt/无翅信号的异位激活先前已被证明对头部发育具有类似的影响，这表明hdl突变体中缺乏抑制Wnt信号的机制。突变的遗传图谱显示hdl编码Tcf/Lef家族成员Tcf3， Tcf3是一种转录因子，通过与辅抑制因子的关联作为Wnt靶基因的抑制因子，或通过与β -连环蛋白的关联作为激活因子。通过显微注射编码野生型Tcf3的合成RNA来挽救hdl突变表型。缺乏β -连环蛋白结合域的Tcf3缺失和嵌合抑制因子Engrailed-Tcf3也拯救了突变表型。相反，嵌合激活子VP16-Tcf3在野生型胚胎中产生无头表型。我们的研究结果表明，在早期原肠形成过程中，Tcf3对Wnt靶点的基础抑制对于决定前神经命运的基因的表达至关重要。先前的研究表明，分泌的Wnt拮抗剂作为头部诱导剂具有关键作用。我们的研究表明，这些Wnt拮抗剂通过维持Tcf3提供的Wnt靶基因的抑制而发挥头部诱导剂的作用。在缺乏这种抑制的情况下，Wnt拮抗剂是无效的头部诱导剂。神经源性突变，精神炸弹（mib），其特征是早期神经元的过度产生。功能分析表明这些突变体在Notch信号通路上存在缺陷。先前的研究表明，Notch信号介导侧抑制，通常限制胚胎中产生的神经元数量。我们发现Notch信号的激活减少了神经源性表型，抑制Notch信号的基因的异位表达模拟了mib表型。位置克隆帮助确定了一个强有力的mib候选。我们正在确定该候选基因中的突变是否导致了mib突变体中的突变表型。我们也在研究Notch在神经板形成中的作用。Notch3在神经胚层原肠胚形成过程中表达。后来它的表达似乎仅限于神经板和神经管内的神经母细胞。我们发现编码Notch3激活形式的RNA在外胚层中的异位表达增加了神经外胚层的大小。然而，在热休克启动子的控制下，在原肠胚形成过程中，Notch3的一种组成型活性形式的持续表达似乎抑制了细胞被纳入神经管。这些观察结果表明，Notch信号最初促进神经外胚层的形成，但后来它限制了神经外胚层内采用神经命运的细胞数量。这些研究共同揭示了Wnt和Notch信号通路在早期神经发生模式中所起的关键作用。这些研究为Tcf3在Wnt信号传导中所起的关键作用提供了遗传证据，说明斑马鱼突变体如何加深了我们对脊椎动物发育过程中广泛使用的信号通路的理解。

Our goal is to identify cellular, molecular and genetic mechanisms that determine how neurons are made in the appropriate number and location in the neural plate. In order to identify such mechanisms we have identified zebrafish mutants with an aberrant pattern of early neurons. Analysis of these mutants is beginning to define mechanisms that are essential for patterning early neurogenesis in the zebrafish embryo. The headless (hdl) mutant was identified by its expanded domain of trigeminal neurons. Expansion of trigeminal neurons in this mutant is accompanied by an expansion of the neural domain at the midbrain-hindbrain boundary (MHB), adjacent to which trigeminal neurons are normally generated. Expansion of neural domains associated with the MHB is at the cost of anterior neural structures like eyes, forebrain and most of the midbrain, which are lost in this mutant. Ectopic activation of Wnt/Wingless signaling during early gastrulation has previously been shown to have similar effects on head development suggesting that there is loss of a mechanism that inhibits Wnt signaling in hdl mutants. Genetic mapping of the mutation revealed that hdl encodes a member of the Tcf/Lef family, Tcf3, a transcription factor that acts as a repressor of Wnt target genes through its association with co-repressors, or as an activator through its association with beta-catenin. The hdl mutant phenotype is rescued by microinjection of synthetic RNA encoding wild-type Tcf3. The mutant phenotype is also rescued by a deletion of Tcf3 that lacks the beta-catenin binding domain, and by a chimaeric repressor, Engrailed-Tcf3. Conversely, a chimaeric activator, VP16-Tcf3, generates a headless phenotype in wild-type embryos. Our results suggest that basal repression of Wnt targets by Tcf3 during early gastrulation is essential for expression of genes responsible for determining anterior neural fate. Previous studies have suggested a critical role for secreted Wnt antagonists as head inducers. Our studies suggest that these Wnt antagonists act as head inducers by maintaining repression of Wnt target genes provided by Tcf3. In the absence of this repression the Wnt antagonists are ineffective head inducers. The neurogenic mutant, mind bomb (mib), is characterized by an over-production of early neurons. Functional analysis suggests these mutants have a defect in the Notch signaling pathway. Previous studies have shown that Notch signaling mediates lateral inhibition and normally limits the number of neurons produced in the embryo. We showed that activation of Notch signaling reduces the neurogenic phenotype and ectopic expression of genes that inhibit Notch signaling mimic the mib phenotype. Positional cloning has helped identify a strong candidate for mib. We are in the process of determining if mutations in this candidate are responsible for the mutant phenotype in mib mutants. We are also investigating the role of Notch in neural plate formation. Notch3 is expressed during gastrulation in the neurectoderm. Later its expression appears to be restricted to neuroblasts within the neural plate and neural tube. We find that ectopic expression of RNA encoding an activated form of Notch3 in the ectoderm increases the size of the neurectoderm. However, persistent expression of a constitutively active form of Notch3 during gastrulation, induced by expressing it under control of a heat shock promoter, appears to inhibit cells from being incorporated in the neural tube. These observations suggest that Notch signaling initially promotes the formation of the neurectoderm but later it limits the number of cells that adopt a neural fate within the neurectoderm.Together these studies reveal critical roles played by the Wnt and Notch signaling pathways in patterning early neurogenesis. By providing genetic evidence for the critical role played by Tcf3 in Wnt signaling these studies illustrate how zebrafish mutants are deepening our understanding of signaling pathways that are widely used during vertebrate development.