WNT control of patterning and cell lineage in limb development

WNT 控制肢体发育中的模式和细胞谱系

• 批准号: RGPIN-2017-04828
• 负责人: Brook, William
• 金额: $ 2.48万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710770
• 关键词: WNT; control; patterning; cell; lineage

We propose to study the mechanism of action of Wnt signalling in pattern formation and cell lineage in the Drosophila melanogaster leg. The expression of the fly Wnt-1 homolog wingless (wg) is localized to the ventral most cells of the leg. Wg acts as a tissue organizer coordinating the development of the ventral quadrant of the leg. Wg is both absolutely necessary and completely sufficient to induce ventral patterns of development. Two other Wnts, Dwnt4 and Dwnt6 are co-expressed with wg in the ventral leg. We propose a series of studies with the goal of resolving and clarifying 1) the relative contribution of the three ventral Wnt genes, 2) the range of action and cellular mechanism employed by Wnts, and 3) the influence of Wnt on ventral cell-lineage mechanisms. In Objective 1 we address a current controversy about whether Wg acts as a morphogen in a long-range direct manner or whether it signals only locally. The controversy arises because in some experiments a membrane-tethered version of Wg (Nrt-wg) is able to rescue Wg function in wing patterning but in other cases it cannot. The difficulties in resolving this conflict stems from the complex expression of Wg and the limited availability of downstream target genes and morphological markers for studying the readout of Wg singling in the wing. We argue that the fly leg offers a better system for asking how Wg signals because the expression is consistently restricted to the organizer region and because of better morphological markers and associated target genes. We will first address whether the other two Wnt molecules compensate for loss of wg. This would provide an explanation for some of the contradictory results described above. We will examine systematically how modulating Wg signaling affects both the gene expression patterns and morphology of the ventral leg and whether loss-or gain-of-function of Wg signaling indicates that the genes are activated in a long-range manner predicted for a morphogen. In Objective 2 we study the role of Wg and Dpp, a fly BMP, in a novel lineage boundary we have identified between Wg-dependent ventral cells and Dpp-dependent dorsal cells. Lineage tracing shows that clones of ventral cells are not recovered in the dorsal domain and vice-versa. We will use lineage tracing of cells compromised for Wg and/or Dpp signaling or downstream target genes to determine which pathway is responsible for the lineage boundary. By observing behaviors of labeled clones and by manipulating known and candidate genes downstream of Wg and Dpp, we will test whether the lineage restriction is controlled by cell competition and cell death, cell adhesion or mechanical tension.

我们建议研究Wnt信号在果蝇腿的图案形成和细胞谱系中的作用机制。苍蝇WNT-1同源基因无翼(WG)的表达定位于腿腹侧大部分细胞。工作组作为一个组织组织者，协调腿部腹侧象限的发育。WG既是绝对必要的，也是完全充分的，可以诱导腹侧发育模式。另外两个WNT4和DWNT6与WG在腹侧腿中共表达。我们提出了一系列的研究，目的是解决和澄清1)腹侧三个WNT基因的相对贡献，2)WNT的作用范围和细胞机制，以及3)WNT对腹侧细胞谱系机制的影响。 在目标1中，我们解决了目前的一个争议，即Wg是以一种远程直接的方式起到形态作用，还是它只在局部发出信号。引起争议的原因是，在一些实验中，Wg的膜拴版本(NRT-WG)能够挽救Wg在翅膀模式中的功能，但在其他情况下，它不能。解决这一冲突的困难源于Wg的复杂表达，以及用于研究Wg在翅膀上的读出的下游靶基因和形态标记的有限。我们认为，苍蝇腿提供了一个更好的系统来询问Wg是如何发出信号的，因为Wg的表达始终限于组织者区域，而且因为有更好的形态标记和相关的靶基因。我们将首先解决另外两个WNT分子是否补偿WG的损失。这将为上述一些相互矛盾的结果提供解释。我们将系统地研究调节Wg信号如何影响腹侧腿的基因表达模式和形态，以及Wg信号的功能丧失或获得是否表明这些基因被以一种预测为形态基因的长期方式激活。 在目标2中，我们研究了Wg和DPP在Wg依赖的腹侧细胞和DPP依赖的背侧细胞之间的一种新的谱系边界中的作用。谱系追踪表明，腹侧细胞的克隆不能在背侧区域恢复，反之亦然。我们将使用Wg和/或DPP信号受损的细胞或下游靶基因的谱系追踪来确定哪条途径负责谱系边界。通过观察标记克隆的行为，以及通过操纵Wg和DPP下游的已知和候选基因，我们将测试谱系限制是否受细胞竞争和细胞死亡、细胞黏附或机械张力的控制。