Cascade regulation of sweat gland development by Wnt, Eda and Shh pathways [beta-catenin knockout]

Millions of sweat glands required to maintain body temperature develop from embryonic ectoderm by a poorly defined mechanism. We present evidence for temporal cascade regulation of sweat gland development by Wnt, Eda and Shh pathways. The first stage, sweat gland induction, failed completely when Wnt/beta-catenin signaling was blocked in skin epithelium, accompanied by sharp downregulation of Wnt, Eda and Shh pathway genes. In a meta-layer of regulation, Wnt antagonist Dkk4 appeared to operate on sweat gland induction in a negative feedback loop: Dkk4 was itself sharply downregulated in beta-catenin-ablated mice, whereas its over-expression repressed Wnt target genes and significantly reduced gland numbers. Eda signaling succeeded Wnt, and activated downstream Shh pathway. Thus, in absence of Eda, Wnt pathway was still active and initial sweat gland pre-germs were seen but failed to develop germs, and dwonstream Shh pathway was repressed. When both Wnt and Eda were intact but Shh was ablated, early stage induction and subsequent duct formation occurred normally, but the final stage formation of secretory coil failed. Thus, sweat gland development shows a relay of regulatory steps, initiated by Wnt/beta-catenin -- itself modulated by Dkk4 -- with subsequent tandem action of Eda and Shh pathways. Compared expression changes between wild type and skin specific beta-catenin knockout footpads at 3 developmental time points, E15.5, E16.5 and E17.5. Data were duplicated with 2 sets of samples.维持体温所需的数百万汗腺，其发育起源于胚胎外胚层，但具体调控机制尚未明确。本研究针对Wnt、Eda及Shh信号通路对汗腺发育的时序级联调控提供了实验证据。第一阶段为汗腺诱导阶段：当皮肤上皮细胞中的Wnt/β-连环蛋白信号通路被阻断时，汗腺诱导过程完全失败，同时Wnt、Eda及Shh通路相关基因的表达水平显著下调。在调控的多层级网络中，Wnt拮抗剂Dkk4通过负反馈环路参与汗腺诱导的调控：β-连环蛋白敲除小鼠体内的Dkk4表达水平显著下调，而Dkk4过表达则会抑制Wnt靶基因的表达，并显著减少腺体生成数量。Eda信号通路的激活晚于Wnt通路，且可激活下游的Shh信号通路。因此，在缺失Eda的情况下，Wnt通路仍可保持激活状态，虽可观察到初始汗腺前体芽基，但无法进一步发育为成熟芽基，且下游Shh通路的表达受到抑制。当Wnt与Eda通路均保持完整，但Shh通路被敲除时，早期诱导及后续导管形成过程可正常进行，但最终分泌性螺旋结构的形成宣告失败。综上，汗腺发育呈现一系列连续的调控步骤：由Wnt/β-连环蛋白信号通路启动（其活性受Dkk4调控），随后依次由Eda与Shh信号通路协同完成后续发育过程。本研究选取三个小鼠胚胎发育时间点（E15.5、E16.5及E17.5），对野生型与皮肤特异性β-连环蛋白敲除小鼠的足垫组织进行了基因表达差异分析，实验设置两组生物学重复样本以保障数据可靠性。