Response to Hedgehog signaling in hair follicle epithelial cells is not associated with short-term hair regeneration

doi:10.3969/j.issn.2095-4344.1779

Abstract

Abstract:

BACKGROUND: Hedgehog signaling plays a vital role in development, homeostasis and tissue repair of multiple tissues. Previous studies underscore that Hedgehog signaling is essential for hair morphogenesis.
OBJECTIVE: To explore the role of Hedgehog signaling in adult epidermal homeostasis.
METHODS: Lgr5-EGFP-Ires-CreER^T², Smo^fl/fl and K14-CreER mice were purchased from The Jackson Laboratory. This study was approved by the Laboratory Animal Ethics and Use Committee of Shanghai Jiao Tong University with the approval No. 1602028. First, Lgr5-EGFP-Ires-CreER^T2 mice were used to specifically label Lgr5⁺ cell subsets, confirming several Hedgehog signaling components were enriched in Lgr5⁺ hair follicle epidermal stem cells/precursor cells. Lgr5-EGFP-Ires-CreER^T2 mice then copulated with Smo^fl/flmice. Lgr5-EGFP-Ires-CreERT2;Smo^fl/fl mice were obtained and shaved of their fur at P49 days of the second telogen period, followed by tamoxifen-induced Hedgehog signaling pathway-mediated Smo knockout. We observed the effect of Smo knockout on hair regeneration rate in Lgr5⁺ cells. In the following experiments, tamoxifen was used to induce Smo knockout on P50-54 days, and the mice were shaved of their fur the mice at 1 week after Smo knockout. We further evaluated the functional changes of bulge epithelial stem cells in mutant mice. (2) K14-CreER mice copulated with Smo^fl/fl to produce K14-CreER;Smo^fl/^fl mice. Tamoxifen, as described above, was used to delete Smo in hair follicle epithelial basal cells. We observed whether hair loss disorder and hair follicle Bulge stem cell dysfunction occur.
RESULTS AND CONCLUSION: No hair loss disorder was detected in Lgr5-EGFP-Ires-CreERT2;Smo^fl/fl and K14-CreER;Smo^fl/fl mouse models, and hair follicle Bulge stem cells were not affected. It is indicated that the responsiveness to Hedgehog signaling in Lgr5⁺ cells and hair follicle epithelial cells is independent of hair follicle telogen-to-anagen transition and short-term hair regeneration as well as the maintenance of Bulge epithelial stem cells. This finding highlights the different molecular mechanisms of tissue development and homeostasis, and provides an important basis for a more accurate understanding of the mechanisms of hair follicle regeneration and tissue growth.

Key words: Hedgehog signaling, Lgr5+ cells, Bulge epithelial stem cells, telogen-to-anagen transition, hair regeneration, Lgr5-EGFP-Ires-CreERT2 mice, Smofl/fl mice, K14-CreER mice, National Natural Science Foundation of China

CLC Number:

R496
Q71

Ying Qinxin, Tao Yixin, Yang Qingchun, Ling Xuejian, Ma Gang. Response to Hedgehog signaling in hair follicle epithelial cells is not associated with short-term hair regeneration[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4044-4049.

Figures/Tables 2

2.1 Lgr5+细胞中高表达Hedgehog信号通路成员为了回答在Lgr5+细胞亚群中的Hedgehog信号对于毛发再生是否必要，利用Lgr5-EGFP-Ires-CreERT2小鼠特异地标记Lgr5+细胞亚群[19]。首先需要确认Hedgehog信号在Lgr5+细胞亚群中的表达情况，因此将6个月大、毛囊处于静息期的对照和Lgr5-EGFP-Ires-CreERT2小鼠的皮肤角化细胞进行流式分选，发现Lgr5+细胞亚群占总角化细胞的0.68%左右(图1A)。通过和总的皮肤角化细胞相比，Lgr5+细胞亚群更丰富地表达bulge上皮干细胞标记物：CD34、Lgr5、Tcf3、Tcf4、Lhx2以及K15(图1B)，这暗示着Lgr5+细胞具有bulge上皮干细胞的一些特性。另外，相对于总的皮肤角化细胞，Lgr5+细胞亚群更丰富地表达Hedgehog信号通路受体Ptch1和Smo，以及Hedgehog信号普适的下游靶基因Gli1、Gli2，但是另外2个Hedgehog靶基因Cyclin D1略上调而Cyclin D2却下调(图1B)。上述结果和前人的报道是一致的[19]，因而验证了在Lgr5+细胞亚群中具有高Hedgehog信号活性。 2.2 Lgr5+细胞对于Hedgehog信号的响应并不影响毛囊静息期-生长期转换在验证了利用Lgr5-EGFP-Ires-CreERT2小鼠特异地标记Lgr5+细胞亚群的可靠性后[19]，实验获得Lgr5-EGFP-Ires-CreERT2;Smofl/fl小鼠，以在Lgr5+细胞亚群中可诱导地敲除Smo，选用母鼠进行接下来所有的实验，以避免激素对于毛发周期的扰动。首先，去观察自然的毛发周期。在刚进入第二轮长静息期的P49天，进行背部剃毛而后连续5 d用他莫昔芬诱导Smo敲除(图2A)。出乎意料的是，在Lgr5+细胞中敲除Smo并不能扰动自然的毛发再生速度(图2B)。为了进一步评估sHG和bulge上皮干细胞在突变小鼠的功能，实验在P50-54天他莫昔芬诱导Smo敲除后1周进行脱毛(图2C)，发现与对照小鼠相比，Lgr5-EGFP-Ires-CreERT2; Smofl/fl小鼠脱毛诱导的静息期-生长期的转换没有差异(图2D)。追踪到脱毛后的16 d也没有发现任何毛发再生过程中的异常(图2E)。脱毛后12 d的皮肤切片苏木精-伊红染色显示Lgr5- EGFP-Ires-CreERT2; Smofl/fl小鼠毛囊进入晚期生长期，也与对照没有明显的差异(图2F)。在他莫昔芬诱导Smo敲除13 d后，Lgr5以及bulge上皮干细胞的标记物K15和CD34的表达与对照相比没有差异(图3)。上述结果说明Lgr5+细胞对于Hedgehog信号的响应对于毛囊静息期-生长期转换并不是必须的，并且对于bulge上皮干细胞的维持也不是必须的。 2.3 毛囊上皮基底层细胞对于Hedgehog信号的响应与毛囊静息期-生长期转换无关上部Bulge上皮干细胞因接收到了感觉神经来源的Shh也显示出Hedgehog信号活性[14]，而这些细胞不表达Lgr5[19]，作者推测是否是上部Bulge上皮干细胞或其他Lgr5-的上皮细胞群在Lgr5-EGFP-Ires-CreERT2; Smofl/fl小鼠中代偿并贡献于毛发再生，而使Lgr5-EGFP-Ires-CreERT2; Smofl/fl小鼠并没有出现毛囊静息期-生长期转换的异常。为此，在更大的细胞群中，即整个毛囊上皮基底层来敲除Smo。实验利用了K14-CreER; Smofl/fl小鼠模型，在刚进入第二轮长静息期的P49天，进行背部剃毛而后连续5 d他莫昔芬诱导，实验结果显示K14-CreER; Smofl/fl小鼠的毛发修复速度和对照也没有显著差异(图4A)。而在P50-54他莫昔芬诱导Smo敲除后1周进行脱毛，也没有发现K14-CreER; Smofl/fl小鼠在脱毛诱导的静息期-生长期的转换过程中有异常(图4B)。上述结果说明毛囊上皮基底层细胞对于Hedgehog信号的响应与毛囊静息期-生长期转换无关。"

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