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Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.

Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S, Watt FM - Cell Stem Cell (2009)

Bottom Line: Lrig1 is a marker of human interfollicular epidermal stem cells and helps maintain stem cell quiescence.Lrig1 is a Myc target gene; loss of Lrig1 increases the proliferative capacity of stem cells in culture and results in epidermal hyperproliferation in vivo.Lrig1-expressing cells can give rise to all of the adult epidermal lineages in skin reconstitution assays.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Epidermal Stem Cell Biology, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK.

ABSTRACT
Lrig1 is a marker of human interfollicular epidermal stem cells and helps maintain stem cell quiescence. We show that, in mouse epidermis, Lrig1 defines the hair follicle junctional zone adjacent to the sebaceous glands and infundibulum. Lrig1 is a Myc target gene; loss of Lrig1 increases the proliferative capacity of stem cells in culture and results in epidermal hyperproliferation in vivo. Lrig1-expressing cells can give rise to all of the adult epidermal lineages in skin reconstitution assays. However, during homeostasis and on retinoic acid stimulation, they are bipotent, contributing to the sebaceous gland and interfollicular epidermis. beta-catenin activation increases the size of the junctional zone compartment, and loss of Lrig1 causes a selective increase in beta-catenin-induced ectopic hair follicle formation in the interfollicular epidermis. Our results suggest that Lrig1-positive cells constitute a previously unidentified reservoir of adult mouse interfollicular epidermal stem cells.

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Effect of Lrig1 Loss on Stem Cell Renewal and Responsiveness to β-Catenin Activation(A) Clonal growth assays of primary keratinocytes sorted based on CD34, Sca1, and α6 integrin (Figure 3A–C) from the skin of wild-type (WT) and Lrig1- (KO) adult littermates. Error bars represent SD (n = 3). Six-hundred Sca1-negative cells and 2500 Sca1-positive and unfractionated cells were seeded; representative dishes are shown.(B and C) Tail epidermal whole mounts of WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. Arrow and insert indicate Lrig1-positive ectopic follicles in interfollicular epidermis.(D and E) Flow cytometric analysis for Lrig1 and α6 integrin of cells from WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. The proportion of cells in each fraction is indicated.(F) Q-PCR of Lrig1 and cMyc mRNA in back skin of WT and K14ΔNβ-cateninER-transgenic mice treated with 4OHT for 2 weeks. Expression levels are relative to Gapdh, and error bars represent SD (n = 3).(G) Q-PCR of levels of Lrig1, Axin2, and Jagged1 in primary murine epidermal keratinocytes treated with Wnt3A. Data are expressed relative to unstimulated cells. Error bars represent SD (n = 3).(H) ChIP analysis of endogenous cMyc on the Lrig1 promoter in WT and K14ΔNβ-cateninER mice treated with 4OHT for 10 days. Data represent two separate samples and show level of isolated genomic DNA relative to amount of input DNA.(I–M) Hematoxylin and eosin stained sections (I and J) and whole mounts (L and M) of adult tail epidermis from K14ΔNβ-cateninER × Lrig1 heterozygous (het) or knockout (KO) mice treated with 4OHT for 3 weeks. The number of ectopic HFs formed from the interfollicular epidermis was scored by morphology (K) and by the appearance of clusters of CDP-expressing cells (N) (two independent experiments; KO, n = 8; WT, n = 7). Arrows in (J) indicate expanded infundibulum with associated ectopic follicles.In (B), (C), (L), and (M), color coding indicates antibody labeling. Scale bars, 100 μm (B and C) and 200 μm (I, J, L, and M).
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fig7: Effect of Lrig1 Loss on Stem Cell Renewal and Responsiveness to β-Catenin Activation(A) Clonal growth assays of primary keratinocytes sorted based on CD34, Sca1, and α6 integrin (Figure 3A–C) from the skin of wild-type (WT) and Lrig1- (KO) adult littermates. Error bars represent SD (n = 3). Six-hundred Sca1-negative cells and 2500 Sca1-positive and unfractionated cells were seeded; representative dishes are shown.(B and C) Tail epidermal whole mounts of WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. Arrow and insert indicate Lrig1-positive ectopic follicles in interfollicular epidermis.(D and E) Flow cytometric analysis for Lrig1 and α6 integrin of cells from WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. The proportion of cells in each fraction is indicated.(F) Q-PCR of Lrig1 and cMyc mRNA in back skin of WT and K14ΔNβ-cateninER-transgenic mice treated with 4OHT for 2 weeks. Expression levels are relative to Gapdh, and error bars represent SD (n = 3).(G) Q-PCR of levels of Lrig1, Axin2, and Jagged1 in primary murine epidermal keratinocytes treated with Wnt3A. Data are expressed relative to unstimulated cells. Error bars represent SD (n = 3).(H) ChIP analysis of endogenous cMyc on the Lrig1 promoter in WT and K14ΔNβ-cateninER mice treated with 4OHT for 10 days. Data represent two separate samples and show level of isolated genomic DNA relative to amount of input DNA.(I–M) Hematoxylin and eosin stained sections (I and J) and whole mounts (L and M) of adult tail epidermis from K14ΔNβ-cateninER × Lrig1 heterozygous (het) or knockout (KO) mice treated with 4OHT for 3 weeks. The number of ectopic HFs formed from the interfollicular epidermis was scored by morphology (K) and by the appearance of clusters of CDP-expressing cells (N) (two independent experiments; KO, n = 8; WT, n = 7). Arrows in (J) indicate expanded infundibulum with associated ectopic follicles.In (B), (C), (L), and (M), color coding indicates antibody labeling. Scale bars, 100 μm (B and C) and 200 μm (I, J, L, and M).

Mentions: To examine the effects of Lrig1 loss on keratinocyte growth in culture, we performed clonal growth assays on the six epidermal populations sorted on the basis of CD34, Sca1, and α6 expression from the back skin of telogen mice (Figure 7A). There was an increase in the colony-forming efficiency of each population and also of total basal cells (all: α6 positive, low forward, and side scatter). This is consistent with expression of Lrig1 in all subpopulations of cells, albeit at different levels (Figure S2E), and with upregulation of cMyc in cultured keratinocytes (Figure 5 and data not shown). However, the increase was most dramatic in the CD34-negative, Sca1-negative, α6 integrin-high population, the cells that normally are highly enriched for Lrig1 expression. We conclude that, in the absence of Lrig1, this population of cells has a selective growth advantage.


Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.

Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S, Watt FM - Cell Stem Cell (2009)

Effect of Lrig1 Loss on Stem Cell Renewal and Responsiveness to β-Catenin Activation(A) Clonal growth assays of primary keratinocytes sorted based on CD34, Sca1, and α6 integrin (Figure 3A–C) from the skin of wild-type (WT) and Lrig1- (KO) adult littermates. Error bars represent SD (n = 3). Six-hundred Sca1-negative cells and 2500 Sca1-positive and unfractionated cells were seeded; representative dishes are shown.(B and C) Tail epidermal whole mounts of WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. Arrow and insert indicate Lrig1-positive ectopic follicles in interfollicular epidermis.(D and E) Flow cytometric analysis for Lrig1 and α6 integrin of cells from WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. The proportion of cells in each fraction is indicated.(F) Q-PCR of Lrig1 and cMyc mRNA in back skin of WT and K14ΔNβ-cateninER-transgenic mice treated with 4OHT for 2 weeks. Expression levels are relative to Gapdh, and error bars represent SD (n = 3).(G) Q-PCR of levels of Lrig1, Axin2, and Jagged1 in primary murine epidermal keratinocytes treated with Wnt3A. Data are expressed relative to unstimulated cells. Error bars represent SD (n = 3).(H) ChIP analysis of endogenous cMyc on the Lrig1 promoter in WT and K14ΔNβ-cateninER mice treated with 4OHT for 10 days. Data represent two separate samples and show level of isolated genomic DNA relative to amount of input DNA.(I–M) Hematoxylin and eosin stained sections (I and J) and whole mounts (L and M) of adult tail epidermis from K14ΔNβ-cateninER × Lrig1 heterozygous (het) or knockout (KO) mice treated with 4OHT for 3 weeks. The number of ectopic HFs formed from the interfollicular epidermis was scored by morphology (K) and by the appearance of clusters of CDP-expressing cells (N) (two independent experiments; KO, n = 8; WT, n = 7). Arrows in (J) indicate expanded infundibulum with associated ectopic follicles.In (B), (C), (L), and (M), color coding indicates antibody labeling. Scale bars, 100 μm (B and C) and 200 μm (I, J, L, and M).
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fig7: Effect of Lrig1 Loss on Stem Cell Renewal and Responsiveness to β-Catenin Activation(A) Clonal growth assays of primary keratinocytes sorted based on CD34, Sca1, and α6 integrin (Figure 3A–C) from the skin of wild-type (WT) and Lrig1- (KO) adult littermates. Error bars represent SD (n = 3). Six-hundred Sca1-negative cells and 2500 Sca1-positive and unfractionated cells were seeded; representative dishes are shown.(B and C) Tail epidermal whole mounts of WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. Arrow and insert indicate Lrig1-positive ectopic follicles in interfollicular epidermis.(D and E) Flow cytometric analysis for Lrig1 and α6 integrin of cells from WT and K14ΔNβ-cateninER mice treated with 4OHT for 3 weeks. The proportion of cells in each fraction is indicated.(F) Q-PCR of Lrig1 and cMyc mRNA in back skin of WT and K14ΔNβ-cateninER-transgenic mice treated with 4OHT for 2 weeks. Expression levels are relative to Gapdh, and error bars represent SD (n = 3).(G) Q-PCR of levels of Lrig1, Axin2, and Jagged1 in primary murine epidermal keratinocytes treated with Wnt3A. Data are expressed relative to unstimulated cells. Error bars represent SD (n = 3).(H) ChIP analysis of endogenous cMyc on the Lrig1 promoter in WT and K14ΔNβ-cateninER mice treated with 4OHT for 10 days. Data represent two separate samples and show level of isolated genomic DNA relative to amount of input DNA.(I–M) Hematoxylin and eosin stained sections (I and J) and whole mounts (L and M) of adult tail epidermis from K14ΔNβ-cateninER × Lrig1 heterozygous (het) or knockout (KO) mice treated with 4OHT for 3 weeks. The number of ectopic HFs formed from the interfollicular epidermis was scored by morphology (K) and by the appearance of clusters of CDP-expressing cells (N) (two independent experiments; KO, n = 8; WT, n = 7). Arrows in (J) indicate expanded infundibulum with associated ectopic follicles.In (B), (C), (L), and (M), color coding indicates antibody labeling. Scale bars, 100 μm (B and C) and 200 μm (I, J, L, and M).
Mentions: To examine the effects of Lrig1 loss on keratinocyte growth in culture, we performed clonal growth assays on the six epidermal populations sorted on the basis of CD34, Sca1, and α6 expression from the back skin of telogen mice (Figure 7A). There was an increase in the colony-forming efficiency of each population and also of total basal cells (all: α6 positive, low forward, and side scatter). This is consistent with expression of Lrig1 in all subpopulations of cells, albeit at different levels (Figure S2E), and with upregulation of cMyc in cultured keratinocytes (Figure 5 and data not shown). However, the increase was most dramatic in the CD34-negative, Sca1-negative, α6 integrin-high population, the cells that normally are highly enriched for Lrig1 expression. We conclude that, in the absence of Lrig1, this population of cells has a selective growth advantage.

Bottom Line: Lrig1 is a marker of human interfollicular epidermal stem cells and helps maintain stem cell quiescence.Lrig1 is a Myc target gene; loss of Lrig1 increases the proliferative capacity of stem cells in culture and results in epidermal hyperproliferation in vivo.Lrig1-expressing cells can give rise to all of the adult epidermal lineages in skin reconstitution assays.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Epidermal Stem Cell Biology, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK.

ABSTRACT
Lrig1 is a marker of human interfollicular epidermal stem cells and helps maintain stem cell quiescence. We show that, in mouse epidermis, Lrig1 defines the hair follicle junctional zone adjacent to the sebaceous glands and infundibulum. Lrig1 is a Myc target gene; loss of Lrig1 increases the proliferative capacity of stem cells in culture and results in epidermal hyperproliferation in vivo. Lrig1-expressing cells can give rise to all of the adult epidermal lineages in skin reconstitution assays. However, during homeostasis and on retinoic acid stimulation, they are bipotent, contributing to the sebaceous gland and interfollicular epidermis. beta-catenin activation increases the size of the junctional zone compartment, and loss of Lrig1 causes a selective increase in beta-catenin-induced ectopic hair follicle formation in the interfollicular epidermis. Our results suggest that Lrig1-positive cells constitute a previously unidentified reservoir of adult mouse interfollicular epidermal stem cells.

Show MeSH
Related in: MedlinePlus