Cellular, Molecular And Genetic Analysis Of Neural Fate

我们的目标是确定神经元在早期神经发生过程中是如何在适当的数量和位置形成的。以前的研究已经在决定果蝇、果蝇和脊椎动物早期发育过程中神经命运的机制上发现了显著的相似之处。在斑马鱼中，神经原蛋白1(Ngn1)的表达定义了细胞在神经板中有可能成为神经元的区域，Ngn1是果蝇神经原基因的同源物。然而，决定Ngn1在神经外胚层早期表达的机制仍然不清楚。易洛魁(Iroquis，Iro)基因编码一类非典型的同源结构域蛋白，最初在果蝇中被发现，因为它们在决定感觉刷毛的形成中起到了作用。对它们在果蝇中的功能分析表明，iro基因早期在确定胚胎中大片区域的身份方面发挥了作用，后来它们在决定神经基因的表达方面发挥了作用。我们在斑马鱼中发现了一个新的iro基因，iro7。在原肠形成过程中，Iro7和Iro1一起在背侧外胚层的一个隔室中表达，该隔室包括预期的中脑-后脑边界(MHB)、相邻的神经脊和三叉神经感觉神经元。Iro1和iro7的表达结构域在无头突变体和主盲突变体中扩展，这些突变体的特征是MHB结构域和邻近组织的扩展，包括形成三叉神经感觉神经元的Ngn1表达结构域。Hdl和mbl突变体中iro1和iro7表达的扩展是由于原肠形成过程中抑制Wnt靶基因的机制的丧失，这表明iro1和iro7的表达区域是由早期发育过程中的Wnt信号决定的。用反义吗啉寡核苷酸下调iro7的功能表明，iro7在形成三叉神经感觉神经元的区域对Ngn1的表达是必不可少的。Iro1和iro7基因的敲除揭示了在MHB中神经脊的发育和峡部组织者的建立中的其他作用。综上所述，这些结果表明，iro1和iro7是建立组织所必需的，这些组织定义了外胚层背侧隔室的身份，这些iro基因在发育早期就在那里表达。神经性突变体Mind Bomb(MIB)的特征是早期神经元过度产生，功能分析表明这些突变体在Notch信号介导的侧向抑制方面存在缺陷，这通常限制了允许在前神经元域内成为神经元的细胞数量。我们发现，抑制Notch1a和Notch5的功能会导致一种非常类似于MIB突变体的表型，在MIB突变体中，前神经元域中早期神经元数量的增加伴随着HER4的表达减少，HER4的表达由Notch信号诱导。定位克隆表明MIB是一个新的基因，其功能在其他模式生物中尚未确定。它编码一种带有环状结构域的蛋白质，这表明它是一种潜在的泛素连接酶。对五个突变等位基因的分析显示，点突变有望导致环区蛋白质截断或关键氨基酸替换。泛素连接酶促进泛素在特定底物蛋白上的加成，通常针对蛋白酶体中的泛素进行破坏和/或改变其在细胞中的功能。我们目前正在研究MIB作为泛素连接酶的功能如何有助于Notch信号在早期神经发生中的效率。我们对MIB突变体的分析也表明，另一个无张力同源基因zath1以及DeltaA和Notch5等神经基因的表达在定义成为侧线系统中感觉毛细胞的细胞方面发挥了作用。在周围神经系统的这一容易接近的部分，能够检查感觉毛细胞的渐进选择，这使得研究与确定早期神经元分布有关的机制变得很有吸引力。总而言之，这些研究说明了斑马鱼细胞、分子和遗传方法的结合是如何揭示脊椎动物胚胎早期神经发生模式的基本机制。

Our goal is to determine how neurons are made in the appropriate number and location during early neurogenesis. Previous studies have identified remarkable similarities in mechanisms that determine neural fate in the fruit fly, Drosophila, and vertebrates during early development. In zebrafish, expression of neurogenin1 (ngn1), a homolog of the Drosophila proneural gene, atonal, defines proneuronal domains where cells have the potential to become neurons in the neural plate. Mechanisms that determine early expression of ngn1 in the neuroectoderm, however, remain poorly defined. Iroquois (Iro) genes encode an atypical class of homeodomain proteins that were originally identified in Drosophila for their role in determining formation of sensory bristles. Analysis of their function in Drosophila revealed an early role for Iro genes in defining the identity of large territories in the embryo where later they have a role in determining expression of proneural genes. We have identified a novel Iro gene, iro7 in zebrafish. Iro7 is expressed during gastrulation along with iro1 in a compartment of the dorsal ectoderm that includes the prospective midbrain-hindbrain boundary (MHB), the adjacent neural crest and the trigeminal sensory neurons. The iro1 and iro7 expression domain is expanded in headless (hdl) and masterblind (mbl) mutants that are characterized by expansion of the MHB domain and adjacent tissues including the domain of ngn1 expression where trigeminal sensory neurons are formed. Expansion of iro1 and iro7 expression in hdl and mbl mutants is due to the loss of mechanisms that repress Wnt target genes during gastrulation suggesting that the territory of iro1 and iro7 expression is determined by Wnt signaling during early development. A knock-down of iro7 function with anti-sense morpholino oligos revealed that iro7 is essential for expression of ngn1 in the domain where trigeminal sensory neurons are formed. A knock-down of both iro1 and iro7 revealed additional roles in neural crest development and establishment of the isthmic organizer at the MHB. Together, these results suggest that iro1 and iro7 are required for establishment of tissues that define the identity of a dorsal compartment of the ectoderm where these Iro genes are expressed early in development. The neurogenic mutant, mind bomb (mib), is characterized by an over-production of early neurons and functional analysis suggests these mutants have a defect in lateral inhibition mediated by Notch signaling, which normally limits the number of cells permitted to become neurons within proneuronal domains. We found that inhibition of Notch1a and Notch5 function leads to a phenotype very similar to that seen in mib mutants where the increase in the number of early neurons within proneuronal domains is accompanied by reduced expression of HER4, a gene whose expression is induced by Notch signaling. Positional cloning revealed mib is a novel gene whose function had not been determined in other model organisms. It encodes a protein with RING domains identifying it as a potential ubiquitin ligase. Analysis of five mutant alleles revealed point mutations that are expected to result in truncated proteins or in critical amino acid substitutions in the RING domain. Ubiquitin ligases facilitate addition of ubiquitin to specific substrate proteins, typically targeting them for destruction in proteosomes and/or modifying their function in the cells. We are currently investigating how function of mib as a ubiquitin ligase contributes to the efficiency of Notch signaling during early neurogenesis. Our analysis of mib mutants has also revealed that expression of another atonal homolog, zath1, and neurogenic genes like DeltaA and Notch5 play a role in defining cells that become sensory hair cells in the lateral line system. The ability to examine progressive selection of sensory hair cells in this easily accessible part of the peripheral nervous system makes it attractive to study mechanisms involved in determining the distribution of early neurons. Together these studies illustrate how a combination of cellular, molecular and genetic approaches in zebrafish are revealing fundamental mechanisms involved in patterning early neurogenesis in the vertebrate embryo.