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“Hair, Hair” to Tbx1 – An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration

 

Adult or tissue-specific stem cells play major roles in tissue regeneration during normal aging and also during disease. However, the complex multi-factor mechanisms which regulate stem cell self-renewal in adult tissues are relatively unknown. To resolve this, researchers from the laboratory of Elaine Fuchs at the Howard Hughes Medical Institute, The Rockefeller University, New York utilised an RNA-interference-based loss-of function screen in hair follicle stem cells (HF-SCs) to better understand the factors which may govern self-renewal and regenerative potential in stem cells (Chen et al).

Purified primary HF-SCs were transduced with a pool of 2035 candidate shRNAs (5 per gene), which includes nuclear proteins and/or transcription factors that are enriched in stem cells compared with their differentiated  progeny (Blainpan et al and Greco et al) in such numbers so that, on average, a stem cell will express a single shRNA. 95% of the initial shRNAs were detected at 24 hours but after five passages, many shRNAs were depleted or enriched, suggesting that the transduced cells had different long-term proliferative potentials. Genes with a potential involvement in HF-SC function were identified as those in which the shRNAs showed no change after one passage of the cultured HF-SC but did change after 5 passages. These genes, potentially involved in long term self renewal of HF-SCs, represented only 3.5% of genes and included genes such as Hmga2 (needed for NSC self renewal (Nishino et al) and Runx1 (which promotes HF-SC proliferation (Osorio et al). shRNA against these genes, as well as another Tbx1, were progressively selected against over time, and so Tbx1, which has been associated with tissue formation in other organs (Aggarwal et aland Chen et al) was further studied. Tbx1 was found to be expressed at a high level in HF-SCs compared to their cellular progeny while, in vivo the developmental expression of Tbx1 mimicked Sox9 and Lhx2 (essential HF-SC transcription factors) and the adult expression pattern resembled CD34. Conditional targeted knock-out of Tbx1 in the skin epithelium in mice, demonstrated that while hair follicle morphogenesis was normal, after shaving the hair, the Tbx1 knock-out caused the hair follicles to remain quiescent for longer before entering an apparently normal hair development cycle.

Depilation, the process of removing an old hair and the niche cells which help to maintain quiescence while leaving around 80% of stem cells (Ito et aland Hsu et al), was then used to study long term tissue regeneration potential. Wild type HF-SCs induced hair regeneration after multiple rounds of depilation, indicating robust self renewal; however, knock out of Tbx1 led to the loss of 70% of stem cells after 5 rounds of depilation. This was associated with the thinning of the hair coat and a reduction in follicle density, as many Tbx1-KO hair follicles were dormant and had lost the stem cell niche. Further, during normal ageing Tbx1-KO mice lost 30% more stem cells than wild type mice, which also lose a certain amount of Tbx1 expressing HF-SCs. BrdU analysis in WT-mice found that following depilation, stem cell proliferation peaked at day three with 70% of cells being BrdU positive, in comparison to Tbx1-KO mice, whose stem cells proliferated slower with only 25% of stem cells being BrdU-positive, a finding corroborated by DNA-content-based cell-cycle analysis. mRNA analysis after depilation found that a high proportion of cell cycle regulators were downregulated more than 1.8 fold in the Tbx1-KO stem cells as compare to the WT stem cells while those genes enriched in Tbx1-KO stem cells indicated a role for BMP signalling, which correlates with previous work showing that over activation of this pathway causes coat thinning with age (Blessing et al). To study if BMP inhibition could reverse Tbx1-KO, beads soaked with a BMP antagonist (noggin) were injected into follicles of plucked hairs (Botchkarev et al and Kulessa et al). Interestingly, this recovered the reduced proliferation rate for Tbx1-KO-stem cells back to levels observed in WT-mice, with the HF-SC pool sustained through the hair cycle, and this ameliorative effect of BMP inhibition on HF-SCs was further shown by the ablation of the BMP receptor BMPR1A.

Overall, this study suggests that Tbx1 is vitally important for the replenishment of HF-SCs during tissue regeneration through the regulation of stem cell self renewal and intersects with BMP signalling to govern the transition between stem cell quiescence and proliferation in hair follicles. The paper also shows the strength of RNAi-screens, and points to their use in other adult stem cell populations which will help us to better understand their basic biology.

 

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