Cellular, Molecular And Genetic Analysis Of Neural Fate In Zebrafish Embryos

斑马鱼胚胎中的神经元是在离散的神经源性区域中产生的，在这些区域中，与斑马鱼神经原基因相关的碱性螺旋环螺旋转录因子的表达使细胞有可能分化为神经元。然而，表达在相邻细胞表面的Notch受体配体Delta和Jagge激活了前体细胞中的Notch受体，阻止了这些区域内的一些前体细胞的早期分化，并允许它们保持或以后与另一种命运分化。 Notch及其配体都是表达在细胞表面的单通道膜蛋白，它们的相互作用使表达配体的细胞能够调节邻近表达Notch的细胞的命运。Notch受体有一个胞内区(NICD)，可以作为转录激活复合体的一部分发挥作用。NICD结构域的释放和移位受Notch胞外片段(NECD)的去除调控。NECD片段在合成Notch受体的过程中被切割，然而，它仍然与Notch剩余的膜结合片段的胞外残基结合，在那里它阻止进入关键的蛋白水解性切割位点。NECD片段的去除使蛋白酶能够顺序地切割Notch受体，首先在细胞外残基中，然后在膜内区域。这导致NICD片段的释放，然后NICD片段可以移位到细胞核，并在与靶基因中的调节位点结合的辅助因子形成复合体后激活靶基因。 NECD片段的去除和Notch的激活是通过与相邻细胞表面的配体结合来促进的。最近的研究表明，一旦Notch配体，如Delta和锯齿状/锯齿状/锯齿状结合在相邻细胞中，与NECD片段结合的配体就会被表达配体的细胞内化。这导致从剩余的膜结合的Notch受体中分离出NECD片段，并允许蛋白水解性切割，通过将NICD片段释放到细胞内来激活Notch。以前我们发现Delta的内吞作用是由一种环状泛素连接酶--心灵炸弹(Mind Bomb，MIB)触发的，它促进了Delta的泛素化。这一过程导致76个氨基酸的蛋白质泛素的单个或多个分子与Delta胞内区的赖氨酸共价连接。泛素招募内吞机制的组件，这些组件反过来促进泛素化Delta的内吞作用。 在这些研究的背景下，尚不清楚Notch配体的泛素化和内吞是否是由它们与邻近细胞中的Notch相互作用触发的，或者是否像Delta一样，Mib介导的Notch配体的内吞作用是一个独立于它们与Notch相互作用发生的结构性过程。我们现在已经证明，与Notch的相互作用在决定两个Delta同系物DeltaD和DeltaA的内吞作用中起着重要作用，但不能确定第三个同系物DeltaC。首先，我们证明了在中枢神经系统中，大多数Notch配体DeltaD位于细胞质小泡中。然而，在MIB突变体中，DeltaD和DeltaA聚集在细胞表面，这表明Mib介导的DeltaD内吞作用在决定Delta的细胞分布中起着重要作用。然后我们发现在Notch变异体中，通过向胚胎注射反义吗啉来减少Notch受体，DeltaD和DeltaA就像在Mib突变体中一样积累在细胞表面，这表明与Notch的相互作用也是DeltaA和DeltaD内吞作用的重要决定因素，但不是DeltaC的决定因素。 Delta具有在相邻细胞表面(反式)和同一细胞表面(顺式)与Notch相互作用的潜力，因此我们随后研究了顺式或反式相互作用对Delta内吞作用是否至关重要。为了解决这一问题，将用吗啡打倒Notch的胚胎中的细胞移植到有Notch的野生型胚胎中，反之，将带有Notch的野生型细胞移植到没有Notch的胚胎中。在这些特定的环境中，移植细胞及其邻近细胞中Delta的细胞分布的变化帮助我们确定了与同一细胞中的Notch(顺式)或与相邻细胞中的Notch(反式)的相互作用是Delta内吞作用的关键。有趣的是，我们的实验表明，与Notch在顺式和反式中的相互作用有助于DeltaD内吞。 如前所述，与反式转录中的Notch相互作用有助于激活相邻细胞中的Notch。相反，人们认为Delta与顺式Notch的相互作用干扰了Notch在细胞内的激活。我们观察到，顺式分子中的Delta-Notch相互作用有助于Delta内吞作用，在顺式分子相互作用之后，配体-受体复合体的内吞作用降低了两者在细胞表面的可获得性。总之，这些观察结果表明，同一细胞内以及相邻细胞之间的Delta和Notch之间的相互作用如何在Notch信号的动态调节中发挥关键作用。

Neurons in zebrafish embryos are generated in discrete neurogenic domains where expression of basic helix loop helix transcription factors, related to the proneural genes in Drososphila, gives cells the potential to differentiate as neurons. However, activation of the Notch receptor in progenitors by its ligands, Delta and Jagged, expressed on the surface of in neighboring cells, prevents early differentiation of some progenitors within these domains and allows them to be maintained or to differentiate later with alternate fates. Both Notch and its ligands are single pass membrane proteins expressed on the cell surface and their interactions allow the ligand-expressing cell to regulate cell fate in the neighboring Notch-expressing cell. The Notch receptor has an intracellular domain (NICD) that can function as part of a transcriptional activator complex. Release of the NICD domain and its translocation to the nucleus is regulated by removal of an extracellular fragment of Notch (NECD). The NECD fragment is cleaved off during synthesis of the Notch receptor, however, it remains bound to the extracellular stub of the remaining membrane bound fragment of Notch where it prevents access to critical proteolytic cleavage sites. Removal of the NECD fragment allows proteases to sequentially cleave the Notch receptor, first in the extracellular stub and then in the intra-membranous domain. This results in release of the NICD fragment, which can then translocate to the nucleus and activate target genes after forming a complex with co-factors that bind regulatory sites in target genes. Removal of the NECD fragment and activation of Notch is facilitated by its binding to ligands on the surface of neighboring cells. Recent studies have suggested that once Notch ligands, like Delta and Jagged/Serrate bind Notch in a neighboring cell, the ligand bound to the NECD fragment is internalized by the ligand-expressing cell. This results in separation of the NECD fragment from the remaining membrane-bound Notch receptor and allows proteolytic cleavages that activate Notch by releasing the NICD fragment into the cell. Previously we showed that endocytosis of Delta is triggered by a RING ubiquitin ligase, Mind bomb (Mib), which facilitates ubiquitylation of Delta. This process results in covalent linkage of a single or multiple molecules of the 76 amino acid protein, Ubiquitin, to lysines in the intracellular domain of Delta. Ubiquitin recruits components of the endocytic machinery and these in turn facilitate endocytosis of ubiquitylated Delta. In the context of these studies it was not clear if ubiquitylation and endocytosis of Notch ligands was triggered by their interaction with Notch in a neighboring cell or whether Mib-mediated endocytosis of Notch ligands, like Delta, is a constitutive process that takes place independent of their interaction with Notch. We have now shown that interactions with Notch have an important role in determining endocytosis of two Delta homologues, DeltaD and DeltaA, but not a third homologue, DeltaC. First we showed that in the central nervous system most of the Notch ligand, DeltaD, is located in cytoplasmic vesicles. However, in mib mutants, DeltaD and DeltaA accumulate on the cell surface suggesting that Mib-mediated DeltaD endocytosis plays an important role in determining the cellular distribution of Delta. We then showed that in Notch morphants, in which Notch receptors are reduced by injecting embryos with anti-sense morpholinos, DeltaD and DeltaA accumulate on the cell surface just as they do in Mib mutants, suggesting that interaction with Notch is also an important determinant of DeltaA and DeltaD endocytosis but not of DeltaC. Delta has the potential to interact with Notch both on the surface of a neighboring cell (in trans) and on the surface of the same cell (in cis) so we then investigated whether interactions in cis or in trans are critical for Delta endocytosis. To address this issue cells from an embryo in which Notch had been knocked-down with morpholinos were transplanted into wild-type embryos with Notch, and conversely wild-type cells with Notch were transplanted into embryos without Notch. Changes in the cellular distribution of Delta in the transplanted cells and in their neighbors in these specific contexts helped us determine if interactions with Notch within the same cell (in cis) or with Notch in a neighboring cell (in trans) were critical for Delta endocytosis. Interestingly, our experiments demonstrated that interactions with Notch both in cis and in trans contribute to DeltaD endocytosis. As discussed earlier, interactions with Notch in trans help activate Notch in a neighboring cell. In contrast, it is thought that interaction of Delta with Notch in cis interferes with Notch activation within that cell. Our observation that Delta-Notch interactions in cis contribute to Delta endocytosis suggests that, following an interaction in cis, endocytosis of the ligand-receptor complex reduces availability of both at the cell surface. Together these observations show how interactions between Delta and Notch within the same cell and between adjacent cells play a critical role in the dynamic regulation of Notch signaling during development.