水平基底细胞上的不动纤毛可以介导嗅上皮再生

嗅上皮由嗅觉感觉神经元（OSNs）、支持细胞(sustentacularcells/SUS)、Bowman’s gland duct cells、球状基底细胞(globose basal cells/GBCs)和水平基底细胞(horizontalbasal cells/HBCs)组成。GBCs和HBCs被认为是嗅上皮的前体细胞或干细胞，具有促进再生的作用。HBCs被证明具有促进嗅上皮再生的作用，然而其具体的调控机制是未知的。

本文中会提到的一些Maker 总结

1.    HBCs上有不动纤毛

作者首次发现HBCs有不动纤毛（primary cilia），OSNs并不是嗅上皮中唯一有纤毛的细胞类型。这些纤毛位于Basal cell和SUS的细胞质间隙，可能在它们之间起通讯作用。

图1 HBCs上有不动纤毛

Figure 1. HBCs possess primary cilia. Immunofluorescence staining was performed on tissue from the olfactory epithelium of 3-to 6-week-old wild-type, Arl13b-EGFPtg, and EGFP-CETN2 mice. A, p63-labeled HBCs possess ARL13B-labeled cilia (inset, magnified image).B, Cilia extend from -tubulin-labeled basal bodies (arrows). Low-magnification view (left) and high-magnification view (right) of ciliated HBC. C, In Arl13b-EGFPtg mice, K5-labeled HBCs possess GFP  cilia(inset,magnified image).  D, HBC cilia labeledwith canonical ciliamarkers AC3 and ARL13B (arrowheads). E, In Arl13b-EGFPtg mice, AC3 labels GFP  cilia (arrowheads). F, p63-labled HBCs possess AC3-labeled cilia. G, In EGFP-CETN2 mice, GFP-expressing basal bodies possess AC3  cilia (arrowheads). H, In Arl13b-EGFPtg mice, GFP  cilia (arrows) project from p63- labeled HBCs into the interstitial space between HBCs and K18-labeled SUS cell endfeet(inset,magnified image). I, In wild-type mice intranasally infected with adenovirus containing GFP, K5-labeled HBCs possess ARL13B-labeled cilia (see arrow) that project into the interstitial space between an HBC and GFP  end foot of a SUS cell (see inset for higher magnified image). 

2.    纤毛只存在于HBCs，不存在于GBCs

由于HBCs和GBCs都是嗅上皮的基底干细胞，作者想知道是否GBCs上也有不动纤毛？实验结果表明纤毛只存在于HBCs上，这暗示着纤毛可能在其中起着特殊的作用。

图2 HBCs是嗅上皮中唯一有纤毛的基底细胞

Figure 2. HBCs are the predominant ciliated olfactory basal stem cell. Immunofluorescence staining was performed in the olfactory epithelium of wild-type mice. A,The canonical GBC marker MASH1 colocalizes with a subset of SEC8  GBCs, while, LSD1 colocalizes with a larger subset of SEC8  GBCs. B, Few SEC8-labeled GBCs possess ARL13B-labeled cilia (see arrows) compared with K5-labeled HBCs (inset,magnified image). Scalebars, 10m. Dashed line, Basementmembrane. †Occasionalmigrating GBC. C, Quantifieddata of SEC8  cells that are eitherMASH1  (N 4) or LSD1  (N  4). D,The percentage of HBCs (N  6) and GBCs (N  6) that possess cilia. 

3.    IFT88的细胞特异性敲除

       为了验证以上的猜想，作者设计了特异性敲除小鼠品系(K5rtTA;tetOCre;Ift88^fl/fl)，可以使HBCs的纤毛丢失但不会影响OSNscila 和嗅上皮的细胞组成。

图3 IFT88的细胞特异性敲除可以使HBCs的纤毛丢失但不会影响OSNs cila

Figure 3. Cell-specific deletion of Ift88 in HBCs results in the loss of HBC cilia with no effects on OSN cilia. Control and iHBC-IFT88 mutant mice (referred to as iHBC-IFT88) were administered a dox-containing diet at P28 for 1 or 4 weeks,and immunofluorescence staining of the olfactory epithelium was performed.A, After 1 week of the dox-containing diet,Cre ispresent in K5-labeled HBCs of mice with K5rtTA and TetOCre alleles. B, After1week of the dox-containing diet, Cre is absent inmice lacking the TetOCre allele. C, After 4 weeks of the dox-containing diet, in control mice but not in iHBC-IFT88 mice, K5-labeled HBCs possess Arl13b-labeled cilia. D, E, Quantified data show that the percentage of HBCs that are ciliated in control mice is significantly reduced by 88.5% in iHBC-IFT88mice,with no significant difference in the number of HBCs permillimeter of OE in control and iHBC-IFT88mice after 4weeks of the dox-containing diet. F, Acet-Tub-labeled cilia are still present inOMP-labeled matureOSNs(see short arrows).

图4 IFT88的细胞特异性敲除可以使HBCs的纤毛丢失但不会影响嗅上皮的细胞组成。

Figure 4. Loss of HBC cilia has no homeostatic effects on the cell composition of the OE. Control and iHBC-IFT88 mice were administered a doxycycline-containing diet at P28 for 8 weeks. A, In
control mice, but not in iHBC-IFT88 mice, K5-labeled HBCs possess ARL13B-labeled cilia (see arrows; inset, magnified image). B, C, Quantified data show a significant loss of ciliated HBCs in iHBC-IFT88mice,with no change in the number of HBCs permillimeter of OE. D, SEC8-labeled GBCs in the OE of control and iHBC-IFT88mice. E, Quantified data show no significant difference in the number of GBCs per millimeter of OE in both groups. F, OMP-labeled mature OSNs in control and iHBC-IFT88 mice. G, Quantified data show no significant difference in the number of mature OSNs per millimeter of OE between both groups.

4.    HBCs中纤毛的丢失导致损伤后嗅上皮细胞再生异常

为了验证HBCs中纤毛的丢失是否会对嗅上皮细胞的再生产生影响，作者使用了一种嗅觉药物methiazole(MMI)，可以特异性的杀死OSNs，SUS，GBCs，但是不会影响HBCs。作者发现经过处理后OSNs，GBCs的细胞数目减少，嗅上皮的厚度减小，但是caspase-3+的细胞数目没有变化，排除由于细胞凋亡的可能。这说明在没有纤毛存在的情况下，HBCs就不能促进嗅上皮的再生。

为了验证以上结果是由于纤毛的功能导致的而不是IFT88的缺失，作者用ARL13B（ARL13B位于纤毛膜上，在纤毛发生中起作用）特异性敲除小鼠对以上结论进行了验证。

图5 HBC纤毛的缺失导致嗅上皮再生异常

Figure 5. Loss of HBC cilia results in the improper regeneration of the OE and loss of TH expression in the OB. Control and iHBC-IFT88 mice were administered a dox-containing diet at P28 for 4 weeks and then were given an intraperitoneal injection of 75 mg/kg methimazole to ablate the OE.Following 8 weeks of recovery, immunofluorescence staining was performed. A, In the OE of control mice, but not of iHBC-IFT88 mice, K5-labeled HBCs possess ARL13B-labeled cilia. B, SEC8-labeled GBCs in the OE of control and iHBC-IFT88 mice. C, D, Quantified data show that the percentage of HBCs that are ciliated in control mice is significantly reduced in iHBC-IFT88 mice with no difference in the number of HBCs per millimeter of OE between both groups. E, Quantified data show a significant reduction in the number of GBCs per millimeter of OE. F, OMP-labeled mature OSNs and Acet-Tub-labeled cilia in the OE of control and iHBC-IFT88 mice. G, Quantified data show a significant reduction in the number of mature OSNs per millimeter of OE. H, A significantly thinner OE in iHBC-IFT88 mice. I, J, No difference in the number of cleaved caspase-3-labeled apoptotic cells was observed in the OE of control and iHBC-IFT88 mice. K, TH expression within glomeruli (dotted circles) in the OBs of control and iHBC-IFT88 mice. L, Quantified data show that the intensity of TH measured in arbitrary units is significantly reduced in the OBs of iHBC-IFT88 mice.

图6 HBCs中Arl13b的缺失导致嗅上皮再生的异常

Figure 6. Loss of Arl13bin HBCs results in the improper regeneration of the OE. Control and iHBC-ARL13Bmicewere administered a doxycycline-containing diet at P28 for 4weeks to induce the deletion of Arl13b.Micewere thengiven an intraperitoneal injection of 75mg/kgmethimazole to ablate the OE andwere allowed 8weeks of recovery. A, K5-labeled HBCs in the OE of iHBC-ARL13B mice do not possess ARL13B-labeled cilia. B, The number of HBCs per millimeter of OE in both control and iHBC-ARL13B mice. C, SEC8-labeled GBCs in the OE of control and iHBC-ARL13B mice. D, Quantified data show a significant reduction in the number of GBCs per millimeter of OE. E, OMP-labeled mature OSNs and Acet-Tub-labeled cilia in the OE of control and iHBC-ARL13B mice. F, Quantified data show asignificant reduction in the number ofmature OSNs per millimeter of OE.

5.    HBC纤毛的缺失导致嗅上皮发育受损

作者进一步发现HBC纤毛缺失后会导致成熟的OSNs的数目减少，这说明在嗅上皮的发育过程中，HBC的纤毛对于神经发生起着重要的作用。

图7 HBC纤毛的缺失导致嗅上皮发育受损

Figure 7. Loss of HBC cilia results in the impaired development of regions in the OE.Control and iHBC-IFT88 mice were treated with a doxycycline-containing diet atE16 for4.5–5weeks and were analyzed at P28 with immunofluorescence staining of the OE. A–F, OMP-labeled mature OSNs in anterior–posterior sections of control and iHBC-IFT88 mice. G, Illustration depicting the location of sections A–Fin a sagittal view of the mouse olfactory organ. H, Magnifiedview of the boxed region in Dwith OMP-labeled mature OSNs and Acet-Tub-labeled cilia in the OE of control and iHBC-IFT88 mice.I, Quantified data show that the number of mature OSNs per millimeter of OE in iHBC-IFT88 mice is significantly reduced specifically in dorsal-lateral and medial regions of the OE,but not inventral regions.

作者首次发现了在嗅上皮中HBCs也有不动纤毛。并且HBCs的纤毛对于嗅上皮的损伤后再生起着重要作用。这暗示着纤毛病（ciliopathies）不仅与OSNs的损伤，还可能与嗅上皮的维持和再生相关。