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，决定神经元是如何在适当的数量和位置的神经板。为了确定这样的机制，我们已经确定了斑马鱼突变体与早期神经元的异常模式。对这些突变体的分析开始确定对斑马鱼胚胎早期神经发生模式至关重要的机制。无头（hdl）突变体是通过其扩大的三叉神经元结构域来鉴定的。三叉神经元在这种突变体中的扩张伴随着中脑-后脑边界（MHB）的神经域的扩张，三叉神经元通常在该边界附近产生。与MHB相关的神经域的扩展是以前部神经结构如眼睛、前脑和大部分中脑为代价的，这些结构在这种突变体中丢失。早期原肠胚形成期间Wnt/Wingless信号传导的异位激活先前已显示对头部发育具有类似的作用，表明在hdl突变体中抑制Wnt信号传导的机制丧失。突变的遗传作图显示hdl编码Tcf/Lef家族的成员Tcf 3，Tcf 3是一种转录因子，通过与辅阻遏物的结合作为Wnt靶基因的阻遏物，或通过与β-连环蛋白的结合作为激活剂。通过显微注射编码野生型Tcf 3的合成RNA拯救hdl突变体表型。突变型表型也通过缺失缺乏β-连环蛋白结合结构域的Tcf 3和嵌合阻遏物Engrailed-Tcf 3来拯救。相反，嵌合激活因子VP 16-Tcf 3在野生型胚胎中产生无头表型。我们的研究结果表明，在原肠胚形成早期Tcf 3对Wnt靶点的基础抑制对于负责决定前神经命运的基因的表达是必不可少的。 以前的研究表明，分泌的Wnt拮抗剂作为头部诱导剂的关键作用。我们的研究表明，这些Wnt拮抗剂通过维持Tcf 3提供的Wnt靶基因的抑制来作为头部诱导剂。在没有这种抑制的情况下，Wnt拮抗剂是无效的头部诱导剂。神经源性突变，思维炸弹（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信号通路中存在缺陷。以前的研究表明，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信号的激活降低了神经原性表型和抑制Notch信号的基因的异位表达，模拟了mib表型。定位克隆已经帮助确定了mib的强有力的候选者。我们正在确定该候选基因的突变是否是mib突变体突变表型的原因。我们还在研究Notch在神经板形成中的作用。Notch 3在神经外胚层的原肠胚形成期间表达。后来，它的表达似乎仅限于神经板和神经管内的成神经细胞。我们发现，异位表达的RNA编码的激活形式的Notch 3的外胚层的神经外胚层的大小增加。然而，在原肠胚形成期间，通过在热休克启动子的控制下表达Notch 3来诱导Notch 3的组成型活性形式的持续表达，似乎抑制细胞被并入神经管中。这些观察结果表明，Notch信号最初促进了神经外胚层的形成，但后来它限制了神经外胚层内接受神经命运的细胞数量。这些研究揭示了Wnt和Notch信号通路在早期神经发生模式中所起的关键作用。通过提供Tcf 3在Wnt信号传导中发挥关键作用的遗传证据，这些研究说明了斑马鱼突变体如何加深我们对脊椎动物发育过程中广泛使用的信号传导途径的理解。

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.