{"id":602169,"date":"2024-05-29T20:00:00","date_gmt":"2024-05-30T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/arid1b-maintains-mesenchymal-stem-cell-quiescence-via-inhibition-of-bcl11b-mediated-non-canonical-activin-signaling-nature-communications\/"},"modified":"2024-05-30T23:15:01","modified_gmt":"2024-05-31T03:15:01","slug":"arid1b-maintains-mesenchymal-stem-cell-quiescence-via-inhibition-of-bcl11b-mediated-non-canonical-activin-signaling-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/arid1b-maintains-mesenchymal-stem-cell-quiescence-via-inhibition-of-bcl11b-mediated-non-canonical-activin-signaling-nature-communications\/","title":{"rendered":"ARID1B maintains mesenchymal stem cell quiescence via inhibition of BCL11B-mediated non-canonical Activin signaling – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
To investigate the role of ARID1B in regulating MSC fate commitment and mesenchymal tissue homeostasis, we evaluated the expression pattern of ARID1B in the proximal region of the mouse incisor. We found that ARID1B is widely expressed in the dental mesenchyme near the NVB where MSCs reside, as well as in odontoblasts, dental pulp cells, and epithelial cells, but less in the TAC region and pre-odontoblasts (Fig. 1a, b<\/a>). Previous study has shown that GLI1+ cells are MSCs surrounding the NVB19<\/a><\/sup>. To find out whether ARID1B is expressed in these GLI1+ MSCs, we co-stained GLI1 and ARID1B and found that ARID1B+ cells in the NVB region overlap with a sub-population of GLI1+ cells (Fig. 1c, d<\/a>). Thus, we hypothesized that ARID1B plays a role in regulating MSC commitment and tissue homeostasis in the adult mouse incisor.<\/p>\n a<\/b>\u2013d<\/b> Immunostaining of ARID1B (red) in control (a<\/b>, b<\/b>) mouse incisor and co-immunostaining of ARID1B (red)\/\u03b2-galactosidase (\u03b2-gal) (green) in Gli1-LacZ<\/i> mouse incisor (c<\/b>, d<\/b>). b<\/b>, d<\/b> represent the high-magnification image of the box in (a<\/b>, c<\/b>). White dotted lines outline the cervical loop. White arrows indicate ARID1B+ cells. Yellow arrows indicate the ARID1B+\/GLI1+ cells. e<\/b>\u2013j<\/b> Notch movement assay in control (e<\/b>\u2013g<\/b>) and Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> (h<\/b>\u2013j<\/b>) mice. Yellow dotted lines show the notch position. Blue lines show the gingival margin. TMX, tamoxifen; wpt, week post-tamoxifen injection. Illustration below (e<\/b>\u2013k<\/b>) created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Internatioanl license. k<\/b> Quantification of the notch movement every other day from D2 to D10. Data are mean\u2009\u00b1\u2009SEM, control n<\/i>\u2009=\u20095, Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> n<\/i>\u2009=\u20097, unpaired two-tailed Student\u2019s t-test. D2, p<\/i>\u2009=\u20090.0083; D4, p<\/i>\u2009=\u20090.0103; D6, p<\/i>\u2009=\u20090.0001; D8, p<\/i>\u2009=\u20090.0003; D10, p<\/i>\u2009=\u20090.0001. l<\/b>\u2013o<\/b> MicroCT and HE staining of control (l<\/b>, m<\/b>) and Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> (n<\/b>, o<\/b>) mouse incisors at 3 months after tamoxifen induction. l<\/b>, n<\/b> MicroCT of control (l<\/b>) and Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> (n<\/b>) mouse incisors. White arrow indicates the narrowed dental pulp. m<\/b>, o<\/b> HE staining of control (m<\/b>) and Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> (o<\/b>) mouse incisors. Yellow arrows indicate the initiation of odontoblast polarization. Green arrow indicates the disordered alignment of odontoblasts. White two-way arrows indicate the dentin thickness. Black asterisk indicates stacked and distorted dentin. Boxes in m and o are shown at higher magnification on the right. p<\/b>, q<\/b> Dspp<\/i> (red) in situ hybridization in control (p<\/b>) and Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> (q<\/b>) mouse incisors. White dotted lines outline the cervical loop. Yellow dotted lines show the cervical loop bending point to the odontoblast initiation distance. Unfilled arrows indicate the distance between the yellow dotted lines. n<\/i>\u2009=\u20093. r<\/b> Quantification of the dental pulp cavity. Data are mean\u2009\u00b1\u2009SEM, n<\/i>\u2009=\u20093, unpaired two-tailed Student\u2019s t-test. p<\/i>\u2009=\u20090.0113. s<\/b> Quantification of the distance of Dspp<\/i>+ cells to the cervical loop. Data are mean\u2009\u00b1\u2009SEM, n<\/i>\u2009=\u20093, unpaired two-tailed Student\u2019s t<\/i>-test. p<\/i>\u2009<\u20090.0001. Source data are provided as a Source Data file. Scale bars: 2\u2009mm (e<\/b>\u2013j<\/b>, l<\/b>, n<\/b>); 100\u2009\u03bcm (other images).<\/p>\n<\/div>\n<\/div>\n GLI1+ MSCs located surrounding the NVB support the mouse incisor\u2019s growth and replenishment throughout the lifespan. Using the Gli1-CreER<\/i> line, we generated Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> mice, in which Arid1b<\/i> was inactivated in the GLI1+ lineage after tamoxifen induction at one month of age, and confirmed that Arid1b<\/i> was effectively deleted from the incisor mesenchymal and epithelial cells (Supplementary Fig. 1a<\/a>\u2013d<\/a>). To evaluate the impact of the loss of Arid1b<\/i> in MSCs on the mouse incisor growth, we first performed an incisor growth assay by comparing the movement of a notch made in the incisor enamel above the gingival margin in both control and Arid1b<\/i> mutant mice. We found that the growth rate was significantly slower in Arid1b<\/i> mutant mice than in the control across all measurement time points (Fig. 1e\u2013k<\/a>). This result indicated that the loss of Arid1b<\/i> in the GLI1+ lineage impairs the adult mouse incisor growth.<\/p>\n Next, we assessed the long-term impact following the loss of Arid1b<\/i>. At 2 months post-tamoxifen induction, the dental pulp cavity was narrower in Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> mice than in the control, as shown by microCT (Supplementary Fig. 1e<\/a>, h<\/a>). Histologically, the polarization of odontoblasts (Supplementary Fig. 1f<\/a>, i<\/a>) and the expression of odontoblast differentiation marker Dspp<\/i> (Supplementary Fig. 1g<\/a>, j<\/a>) were initiated more proximally to the cervical loop in Arid1b<\/i> mutant mice than in the control mice. The cervical loop also appeared to be smaller in the Arid1b<\/i> mutant mice. The odontoblast alignment was affected, and the dentin appeared thicker in the Arid1b<\/i> mutant mice. Moreover, the phenotype of the Arid1b<\/i> mutant mice became more severe at 3 months post-tamoxifen induction, with a limited dental pulp cavity and stacked dentin at the proximal end of the incisor (Fig. 1l\u2013o, r<\/a>). The odontoblasts were well aligned, and their differentiation was marked by the organized alignment of nuclei along the basement membrane in the control; in contrast, the organization of the odontoblasts was abnormal, and the nuclei were in the opposite position in Arid1b<\/i> mutant mice. The odontoblasts were premature in the proximal region in the Arid1b<\/i> mutant mice, as confirmed by Dspp<\/i> marker staining (Fig. 1p, q, s<\/a>). These results indicated that ARID1B plays a role in maintaining adult mouse incisor tissue homeostasis.<\/p>\n Furthermore, to investigate the cellular changes underlying the reduced growth rate and abnormal dentin formation in Arid1b<\/i> mutant incisors, we evaluated the potential of TACs to differentiate into odontoblasts after the loss of Arid1b<\/i>. We labeled TACs in the DNA synthesis phase using EdU injection and harvested the tissue 48\u2009h later to assess the TAC differentiation. The overlap between Dspp<\/i>+ odontoblasts and EdU-labeled cells represented the TAC differentiation ability, and the overlap length indicated the migration rate of these differentiated cells during the preceding 48\u2009h. We observed a reduced overlap length of EdU+\/Dspp<\/i>+ cells in Arid1b<\/i> mutant mice compared to controls at 1 week post-induction, indicating that loss of Arid1b<\/i> caused the compromised migration rate of the differentiated TACs (Supplementary Fig. 1k<\/a>\u2013n<\/a>, q<\/a>). To understand the cause of abnormal dentin formation, we administered calcein and alizarin red S dual fluorescence injections at different time points to dynamically compare the odontoblast migration rate and dentin deposition rate. Using this approach, the fluorescence precipitation represents dentin formation at the time of injection. We compared the odontoblast migration length and dentin deposition depth over a period of 5 days. Statistical analysis revealed a significantly shorter odontoblast migration length in Arid1b<\/i> mutant mice compared to controls, while there was no significant difference in the depth of dentin deposition (Supplementary Fig. 1o<\/a>\u2013p<\/a>, r<\/a>). These findings indicated that the loss of Arid1b<\/i> impairs the odontoblast migration rate, leading to abnormal stacked dentin.<\/p>\n GLI1+ cells contribute to the mesenchymal and epithelial lineages in the mouse incisor19<\/a><\/sup> and ARID1B was effectively deleted in both tissues in the Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> mice. To clarify whether the loss of Arid1b<\/i> in the dental epithelium has any effect on incisor tissue homeostasis, we used the Sox2-CreER<\/i> line to specifically delete Arid1b<\/i> in the epithelial lineage by generating Sox2-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> mice. Again, we induced Cre activity with tamoxifen at 1 month of age and harvested the incisor at 2 months post-induction. There were no signs of either premature odontoblasts or a differentiation defect in Sox2-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> mice as compared to the control (Supplementary Fig. 2a<\/a>\u2013f<\/a>). This result indicated that the loss of Arid1b<\/i> specifically in the dental mesenchyme impairs the incisor growth and tissue homeostasis.<\/p>\n The continuous growth of the mouse incisor is induced by the progression of MSCs giving rise to TACs, along with proliferation and differentiation in the proximal region. Due to the overlap between ARID1B+ cells and a subpopulation of GLI1+ cells in the proximal region, we investigated the potential impact of Arid1b<\/i> loss on GLI1+ MSCs. We generated Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup>;Gli1-LacZ<\/i> mice to compare the numbers of GLI1+ cells in the incisors of these mutants and Gli1-LacZ<\/i> control mice. At 5 days post-induction, the number of GLI1+ cells was significantly reduced in the Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup>;Gli1-LacZ<\/i> mice compared to Gli1-LacZ<\/i> mice (Fig. 2a\u2013e<\/a>). These MSCs are quiescent cells that undergo slow-cycling self-renewal and reside in the proximal region of the mouse incisor. Thus, we investigated the impact of ARID1B on MSCs\u2019 quiescence based on their label-retaining ability. Since incisor mesenchyme turnover takes about 1 month19<\/a><\/sup>, we injected control and Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> mice with EdU for a 1-month period beginning from postnatal day 5 and analyzed the cells after another month. In this case, the label retaining cells (LRCs) detected by EdU staining would be the quiescent cells. We found the number of LRCs was significantly reduced in Gli1-CreER;Arid1b<\/i>fl\/fl<\/i><\/sup> mice compared to controls (Fig. 2f\u2013j<\/a>), confirming that loss of Arid1b<\/i> impairs mouse incisor MSC quiescence.<\/p>\n<\/a><\/div>\n
Loss of Arid1b<\/i> disturbs MSCs\u2019 quiescence and leads to their proliferation<\/h3>\n